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Keywords:

  • Cryptococcus gattii;
  • genotype;
  • Vancouver Island outbreak;
  • VGII;
  • virulence

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Clin Microbiol Infect 2011; 17: 251–258

Abstract

The ongoing cryptococcosis outbreak on Vancouver Island, BC, Canada, is caused by two VGII sub-genotypes of the primary pathogen, Cryptococcus gattii: VGIIa isolates predominate, whereas VGIIb isolates are rare. Although higher virulence of the VGIIa genotype has been proposed, an unresolved key question is whether VGIIa isolates from other regions are also more virulent than VGIIb isolates. We report the relationship between genotype and virulence for a global collection of C. gattii VGIIa and VGIIb isolates (from Australia, Argentina, Brazil, Canada, Thailand and the USA). In vitro and in vivo virulence studies were conducted. At 37°C, growth [at 18 h: 0.2 optical density (OD) difference, p 0.026; at 36 h: 0.6 OD difference, p 0.036) and mean melanin production (OD = 0.25 vs. OD = 0.15, p 0.059] of VGIIa isolates was greater than that of VGIIb isolates. The inhibitory effect of high temperature on melanin production of VGIIa isolates was less than that of VGIIb isolates (OD = 0.36 vs. OD = 0.69; p 0.001). Capsule production at 37°C of VGIIa isolates was less than that of VGIIb isolates. All VGIIa isolates were fertile, whereas only 17% of VGIIb isolates were fertile (p <0.001). In vivo virulence studies using the BALB/c mice nasal inhalation model revealed that VGIIa isolates were more virulent than VGIIb isolates (p <0.001) independent of their clinical (p 0.003) or environmental origin (p <0.001). This study established a clear association between genotype and virulence of the primary fungal pathogen, C. gattii.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

The basidiomycetous yeasts of the Cryptococcus neoformans/Cryptococcus gattii species complex are the most common cause of fatal fungal meningitis in humans and can also cause infections in a wide range of mammals [1]. Two species have been defined: C. neoformans, an opportunistic pathogen, and C. gattii, a primary pathogen [2]. Over the years, several typing methods have been applied to investigate the epidemiology of these pathogenic yeasts. Using serotyping and molecular typing based on M13 fingerprinting, amplified fragment length polymorphism (AFLP) analysis and, more recently, multi-locus sequence typing (MLST) using seven unlinked genetic loci (CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1), four serotypes and eight molecular types have been recognized (C. neoformans: serotype A/molecular types VNI-VNII/AFLP1, D/VNIV/AFLP2, hybrid AD/VNIII/AFLP3; C. gattii: B or C/VGI-IV/AFLP4-7) [3–5]. In addition, interspecies hybrids between serotype B and D have been reported (VNIV and VGI/AFLP2 and AFLP4) [6].

In the microbial world, associations between biotype/genotype and virulence are becoming better characterized. For example, in bacteria, a correlation between the phylogenetic group B1 and Shiga toxin-producing Escherichia coli has been reported [7]. In the case of pathogenic fungi, an epidemiological survey of Candida albicans found a possible association between a specific MLST clade and disease at specific human body sites [8]. However, a direct association between virulence and genotype has never been reported for any medical fungus. In an initial investigation of the cryptococcosis outbreak on Vancouver Island, BC, Canada, it was proposed that the major genotype (VGIIa) was significantly more virulent than the minor genotype (VGIIb), based on virulence studies conducted on a single strain of each genotype [9]. A more recent study by Ma et al. [10] has emphasized and extended this finding by comparing five North American (three from Vancouver Island, BC, Canada, and two from the west coast of the USA) and one Caribbean VGIIa isolate and a single Canadian VGIIb isolate to a global selection of strains representing different major molecular types of the C. neoformans/C. gattii species complex [10]. However, differences in virulence between the two Vancouver Island outbreak genotypes (VGIIa vs. VGIIb) have never been proven systematically using a range of isolates of these genotypes on a global scale. Taking into account the current high human mobility and the associated risk of a spread of human fungal pathogens, it is of critical importance to correlate genotype and virulence on a global scale to predict potential outbreaks and initiate appropriate public health responses [11]. For this reason, we investigated the association between specific genotypes and virulence based on in vitro and in vivo assays using a global selection of VGIIa and VGIIb isolates. The results obtained showed that VGIIa isolates were more virulent than VGIIb isolates, irrespective of their geographic, clinical or environmental origin.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Isolates and growth conditions

Twenty-one C. gattii VGIIa and VGIIb mating type α isolates obtained from Australia, Argentina, Brazil, Canada, Thailand and the USA, as well as the isolate B4546 (clinical VGIII mating type a, used as an opposite mating type strain) from the USA [12], were retrieved from the freeze-dried culture collection of the Molecular Mycology Research Laboratory, Westmead Hospital (Table 1). The original Vancouver Island outbreak strains R265 (VGIIa) and R272 (VGIIb) [9] have been used as reference strains in this study. The molecular types were identified by PCR fingerprinting [4], AFLP analysis [13] and MLST [3]. Each isolate was initially grown on YPD agar (2% glucose, 2% peptone, 1% yeast extract, 2% agar) at 30°C and then subjected to the studies described below. For the experiments using certain CFU/mL as starting points, the C. gattii isolates were initially grown for 24 h in YPD broth at 30°C, washed twice with phosphate buffered saline (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, pH 7.4) and subjected to subsequent studies.

Table 1.   List of isolates and strain characteristics used in the present study
NameCountrySourceMating typeMajor molecular type/genotypeMedian days of survivalFertilityReference
  1. NA, not applicable; N, infertile; F, fertile.

  2. aOriginally studied virulent VGIIa and avirulent VGIIb strains [9].

  3. bA Brazilian person living in Japan.

  4. cVGIII mating type a strain.

R265aCanadaClinicalαVGIIa/AFLP6A31Fertile[13]
F3179CanadaClinicalαVGIIa/AFLP6A29Fertile[13]
RB39CanadaEnvironmentαVGIIa/AFLP6A29Fertile[13]
RB45CanadaEnvironmentαVGIIa/AFLP6A29Fertile[13]
RB59CanadaEnvironmentαVGIIa/AFLP6A32Fertile[13]
CBS7750USAEnvironmentαVGIIa/AFLP6A>50Fertile[32]
NIH444USAClinicalαVGIIa/AFLP6A46Fertile[33]
F1623Brazil/JapanbClinicalαVGIIa/AFLP6A40Fertile[15]
LA295ArgentinaClinicalαVGIIa/AFLP6A26Fertile[4]
R272aCanadaClinicalαVGIIb/AFLP6B>50Fertile[13]
RB28CanadaEnvironmentαVGIIb/AFLP6B>50Infertile[13]
RB31CanadaEnvironmentαVGIIb/AFLP6B>50Infertile[13]
ARN001AustraliaEnvironmentαVGIIb/AFLP6B>50Infertile[34]
RAM002AustraliaEnvironmentαVGIIb/AFLP6B>50Infertile[34]
RAM005AustraliaEnvironmentαVGIIb/AFLP6B>50Infertile[34]
RAM015AustraliaEnvironmentαVGIIb/AFLP6B>50Infertile[34]
McBrideAustraliaVeterinaryαVGIIb/AFLP6B>50Infertile[21]
BandiagaAustraliaClinicalαVGIIb/AFLP6B33Infertile[34]
DMST20765ThailandClinicalαVGIIb/AFLP6B>50Infertile[35]
DMST20767ThailandClinicalαVGIIb/AFLP6B36Infertile[35]
47-5061ThailandClinicalαVGIIb/AFLP6B40Fertile[36]
B4546cUSAClinicalaVGIII/AFLP5NAFertile[12]

In vitro virulence study

To determine the virulence of all isolates, we selected: (i) growth at 37°C; (ii) melanin synthesis; and (iii) capsule size from the set of established cryptococcal virulence factors. All in vitro virulence studies were presented as average values for either the VGIIa or VGIIb isolates studied.

Growth test at 37°C

Each isolate suspension was adjusted to an optical density (OD) of 0.01 (approximately 105 CFU/mL) with 2% glucose YNB and incubated at 37°C. Cell density was determined by spectrophotometry using the OD600 at 0, 18, 36 and 72 h.

Melanin synthesis test

Melanin production was quantified as described previously [14,15]. Supernatants of the cultures were taken at 48 h and their OD at 475 nm was measured. To eliminate growth bias, the cell concentration was determined at 48 h and used to normalize the OD.

Capsule production test

Capsule formation of each isolate was induced under the following capsule-inducing condition: Roswell Park Memorial Institute Media # 1640 (RPMI-1640) with MOPS, HCO3, pH 7.3, in 5% CO2 at 37°C, as described previously [16]. The capsule size of at least 30 yeast cells per studied isolate was measured by light microscopy using the diameter ratio of capsule to capsule : cell wall to cell wall boundary of each cell.

Mating test

Mating assays with the opposite mating type, using strain B4546 (clinical, VGIII, MATa) [12], were carried out on V8 medium [5% (v/v) V8 juice, 2 mM KH2PO4, 4% (w/v) agar] at room temperature in darkness as described previously [17].

In vivo virulence study

Animal virulence studies were conducted with a total of nine VGIIa and twelve VGIIb isolates (Table 1). For each isolate, groups of five immunocompetent female BALB/c mice aged 6 weeks, 18–20 g, (Animal Resources Center, WA, Australia) were infected intranasally with 105 cryptococcal cells as described previously [9].

Statistical analysis

All statistical analyses, including the t-test, chi-square test and Kaplan–Meier survival test, were conducted using spss, version 15.0 (SPSS Inc., Chicago, IL, USA) and blotted using graphpad prism version 5.0b (GraphPad Software Inc., San Diego, CA, USA). p <0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

Strain characteristics

Of the 21 C. gattii VGII isolates studied, nine were genotype VGIIa and 12 isolates were VGIIb. The VGIIa isolates originated from Argentina, Canada, Brazil, Japan and the USA. The VGIIb isolates originated from Australia, Canada and Thailand. Both VGIIa and VGIIb isolates were only obtained together from the same location from Vancouver Island, Canada (Table 1). Within either the VGIIa or VGIIb genotype, all isolates studied to date had M13 PCR fingerprinting and AFLP patterns (Table 1), as well as MLST types identical to those of the reference stains R265 (VGIIa) and R272 (VGIIb), respectively (ST20 for the VGIIa isolates with GenBank accession no. CAP59-GU079812; GPD1-GU079844; IGS1-GU079875; LAC1-GU079906; PLB1-GU079694; SOD1-GU079725; URA5-GU079768; and ST7 for the VGIIb isolates with GenBank accession no. CAP59-GU079817; GPD1-GU079849; IGS1-GU079880; LAC1-GU079911; PLB1-GU079699; SOD1-GU079730; URA5-GU079773).

In vitro phenotypic studies

To define a correlation between genotype and virulence characteristics of the C. gattii VGII isolates, the ability to express the major cryptococcal virulence factors, growth at human body temperature (37°C), melanin synthesis and capsule production, as well as the mating potential, were all compared.

Growth at 37°C

Because of large variation of the starting time point of the log phase among isolates, which ranged from <18 h to over 36 h (data not shown), comparing the doubling time, which is traditionally taken only from log phase data, would be considered inappropriate.

Thus, we compared the overall growth of each isolate based on the fact that concentrations (OD600) of all isolates were equal at time point zero. The VGIIa genotype possessed a greater ability to grow at 37°C in 2% glucose YNB as measured at 18 and 36 h (at 18 h: 0.2 OD difference, p 0.026; at 36 h: 0.6 OD difference, p 0.036) (Fig. 1). Growth difference at 72 h was not statistically significant (p 0.057).

image

Figure 1.  Average growth rates at 37°C in 2% glucose YNB of nine VGIIa and 12 VGIIb isolates (error bars ± SE = 2 SE). OD, optical density.

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Melanin synthesis

At 30°C, VGIIa isolates produced less melanin than the VGIIb isolates (VGIIa OD = 0.61, VGIIb OD = 0.84; p 0.035). Because melanin synthesis might be inhibited at human physiological temperature [18], we also performed the melanin quantification at 37°C.

Interestingly, the opposite finding was observed at mammalian body temperature. Despite weak statistical support (VGIIa OD = 0.25, VGIIb OD = 0.15; p 0.059) for the difference in melanin production between the two genotypes, an inhibitory effect of 37°C on melanin production by VGIIa isolates was less than that on melanin production by VGIIb isolates (VGIIa OD = 0.36, VGIIb OD = 0.69; p 0.001) (Fig. 2).

image

Figure 2.  Graph showing the average melanin production using dopamine as a substrate of nine VGIIa and 12 VGIIb isolates. At 48 h, VGIIa isolates produced less melanin at 30°C (p 0.035), but more melanin at 37°C (p 0.059) (error bars ± SE = 2 SE). OD, optical density.

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Capsule production

Under capsule-inducing conditions, RPMI with MOPS, HCO3 (pH 7.3) in 5% CO2 at 37°C [19], the VGIIa isolates produced a smaller capsule (capsule–capsule : cell wall–cell wall = 2.3) than the VGIIb isolates (capsule–capsule : cell wall–cell wall = 2.6) (p <0.001) (Fig. 3).

image

Figure 3.  Capsule size examples of VGIIa and VGIIb isolates and graph representing the average capsule–capsule : cell wall–cell wall ratio of the nine VGIIa isolates and 12 VGIIb isolates showing that the capsule size of VGIIa is less than that of the VGIIb isolates (×400, p <0.001) (error bars = 2 SE).

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Fertility

All nine VGIIa but only two of the 12 VGIIb isolates were fertile (p <0.001; Table 1). This suggests that the VGIIa genotype has a higher intrinsic ability to undergo sexual reproduction than the VGIIb genotype. There was no difference in the morphology of the teleomorphs between the fertile strains of both genotypes. All fertile isolates of both genotypes produced classical bacillus-shaped basidiospores.

In vivo virulence studies

Overall, VGIIa isolates showed greater virulence than the VGIIb isolates (p <0.001; Fig. 4a–d). As in the immunocompromised mice model used previously [9], the VGIIb strain R272 demonstrated a much lower virulence in the immunocompetent mice model used. This finding was mirrored by the majority of the other VGIIb isolates investigated (Fig. 4d, Table 1). Interestingly, variations in virulence were observed for all investigated isolates (Fig. 4c,d, Table 1) despite the fact that all VGIIa and VGIIb isolates within each group had identical molecular genotypes (data not shown). Clinical and environmental VGIIa isolates (Fig. 4b) were more virulent than clinical and environmental VGIIb isolates (p 0.003 and p <0.001, respectively) (Fig. 4b). For both genotypes, the virulence of clinical isolates was higher than that of environmental isolates, although the difference was only significant for the VGIIb group (p 0.110 for VGIIa isolates; p <0.001 for VGIIb isolates) (Fig. 4c,d).

image

Figure 4.  Murine inhalation model using immunocompetent BALB/c mice. (a) Showing that the VGIIa genotype was more virulent than the VGIIb genotype (p <0.001). (b) Showing the higher virulence of the VGIIa genotype in either a clinical (p 0.003*) or environmental (p <0.001) background. *The veterinary strain McBride was excluded from this analysis because only one veterinary isolate was studied. (c) Individual survival curves for each of the VGIIa isolates studied. (d) Individual survival curves for each of the VGIIb isolates studied. Continuous lines represent VGIIa isolates and dotted lines represent VGIIb isolates in (a) and (b). Continuous lines represent clinical isolates and dotted lines represent environmental isolates in (c) and (d).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

The first extensive outbreak of C. gattii infection occurred in healthy human hosts and a variety of animals on Vancouver Island, BC, Canada, a temperate climatic region [9,13]. Earlier documented outbreaks occurred in goats in Spain [20] and sheep in Western Australia [21]. These outbreaks have emphasized the importance of understanding the epidemiology, pathogenesis and virulence of the primary pathogen, C. gattii.

Intensive epidemiological screening on Vancouver Island and surrounding areas has revealed that approximately 90% of all recovered isolates belonged to the VGIIa genotype, with only approximately 10% belonging to the VGIIb genotype [9,13]. The predominance of the VGIIa genotype raised the possibility that it has a higher level of ‘ecological fitness’ than the VGIIb genotype, in addition to being more virulent in mammalian hosts. Indeed, comparative virulence studies in immunosuppressed A/Jcr mice showed that a representative clinical isolate of the major genotype (strain R265) was more virulent than a representative isolate of the minor genotype (strain R272) [9].

This initial finding raised two major questions. First, was the use of an immunocompromised mice model appropriate for this primary pathogen, which normally causes diseases in immunocompetent hosts? Second, do the results obtained from a single isolate each of the VGIIa and the VGIIb type represent the virulence potential of a representative collection of VGIIa and VGIIb isolates from around the world?

To answer these questions, we investigated and compared the in vitro and in vivo virulence of VGIIa and VGIIb isolates from global sources. The fact that all studied VGIIa isolates possessed a greater ability to grow and produce melanin at the human physiological temperature than the VGIIb isolates (Figs 1 and 2) suggests that the high tolerance to this temperature is a key feature for increased virulence. This is supported by the higher inhibitory effect of 37°C on melanin production of the VGIIb isolates. In addition, the smaller polysaccharide capsule size observed for VGIIa strains (Fig. 3) reflects a finding of an earlier study, showing in vitro evidence for a relationship between capsule size and virulence [22]. Finally, the high fertility of the VGIIa isolates is consistent with the unusually high levels of asexually produced basidiospores or blastoconidia in the environment of Vancouver Island, where most isolates obtained via air sampling belonged to the VGIIa genotype [13]. This in turn provides more infectious propagules, leading to a higher exposure and, ultimately, to a higher infection rate.

In addition to the aforementioned differences in in vitro virulence features, VGIIa isolates were in general more virulent in vivo than VGIIb isolates (p <0.001) in the murine inhalation model using immunocompetent female BALB/c mice. This confirmed the previous suggestion, using immunocompromised A/Jcr mice and only one isolate per VGIIa and VGIIb genotype [9], that the VGIIa genotype is more virulent and showed that this observation is true for a representative range of VGIIa isolates (Fig. 4, Table 1). It also confirms the findings of a later study that emphasized the higher virulence of five North American VGIIa isolates in comparison with isolates from the other major molecular types of the C. neoformans/C. gattii species complex, whereas, again, only the reference VGIIb isolate (R272) was tested [10]. Furthermore, the higher virulence of clinical isolates of both sub-genotypes compared to the environmental isolates is in agreement with recently published data on isolates of the molecular type VNI of the closely related species C. neoformans var. grubii, which showed that clinical isolates were more virulent than environmental isolates using an identical model [23]. They disagree, however, with a previous report reporting greater virulence of environmental isolates of C. gattii [24]. This discrepancy is perhaps the result of only six isolates being tested and the molecular types not being determined [24]. Interestingly, one of those strains, a virulent strain (B4534) in the original studies [24] is the same strain as CBS7750 [25], which showed a low virulence in our study as well as in a recent study by Ma et al. [10] using intranasal or tail vein injection, respectively. The difference in virulence observed in our study could be a result of the different route of infection, implying that lung protection is an important factor for preventing cryptococcosis, although the pulmonary immune response is known to be suppressed by C. gattii [26]. However, Ma et al. [10] used the same route of infection (i.e. intravenous injection) in albino male BALB/c mice of 20–23 g weight, instead of female BALB/c mice of 19–20 g weight, as employed in the original studies by Kwon-Chung et al. [24], but also showed that the strain CBS7750 is a low virulent strain. This may either reflect a different susceptibility to the infection of the mouse strain used or indicate that this specific cryptococcal strain has lost its original virulence as a result of long-term sub-culturing, as was also observed for other cryptococcal strains [27]. Contradicting results obtained from different sets of isolates suggest that the virulence of environmental isolates of different molecular types may differ to a certain extent. Clearly, an extensive virulence survey of additional clinical and environmental C. gattii isolates representing all molecular types is warranted.

In addition to the differences in virulence observed between VGIIa and VGIIb isolates in general and between clinical/environmental isolates of both genotypes specifically, variations in the virulence of individual isolates were observed despite the fact that all isolates within each of the two genotypes had identical M13 PCR fingerprints, AFLP profiles [13] and MLST sequence types (ST20 or ST7, respectively), an observation that had also been reported in a study of the virulence of C. neoformans var. grubii VNI isolates [23]. Taken together, these findings emphasize that several isolates per genotype must be tested before any conclusion can be drawing concerning the virulence of a species, molecular type or genotype within the C. neoformans/C. gattii species complex.

Genotype and virulence have been correlated in a number of pathogenic microorganisms [7,28], including a limited number of C. gattii strains [3,10]. This is the first study to establish a correlation between specific genotypes and virulence for a large number of globally collected VGIIa and VGIIb strains. Although lethal fungal infections are not as frequent as viral and bacterial infections and are mostly limited to immunocompromised patients, small outbreaks of mycoses have periodically emerged in several parts of the world [29–31]. Increasing human mobility and the increasing number of global reports of fungal infection emphasize the need for more surveillance regarding ‘hidden’ potentially highly pathogenic fungal genotypes. Determination of specific genotypes and their correlations with virulence is a vital epidemiological tool for precise and efficient surveillance. Such knowledge is a prerequisite for an appropriate and timely response to newly emerging and highly pathogenic genotypes, in an effort to effectively constrain or even prevent phenomena such as the ongoing cryptococcosis outbreak on Vancouver Island, Canada, or elsewhere.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

We would like to thank M. Krockenberger and A. Harun, University of Sydney, Australia for assistance in this study; J. Kronstad, University of British Columbia, BC, Canada; V. Robert, Centraalbureau voor Schimmelcultures–Fungal Biodiversity Center, the Netherlands; G. Davel, Instituto Nacional de Enfermedades Infecciosas ‘Dr Carlos Gregorio Malbrán’, Argentina; Y. Mikami, Chiba University, Japan, and N. Poonwan, National Institute of Health, Thailand for providing the isolates used in the experiments. None of the authors have any conflict of interest.

Transparency Declaration

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References

This work was supported by an EIPRS and EIPA scholarship of the University of Sydney to PN and a bridging grant (#100124681) of the University of Sydney to WM. RM is supported by the Valentine Charlton Bequest.

References

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Transparency Declaration
  9. References
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