Leona T. Campbell

Clonality and Recombination in Genetically Differentiated Subgroups of Cryptococcus gattii

ABSTRACT Cryptococcus gattii is a pathogenic yeast that together with Cryptococcus neoformans causes cryptococcosis in humans and animals. High numbers of viable C. gattii propagules can be obtained from certain species of Australian Eucalyptus camaldulensis trees, and an epidemiological link between Eucalyptus colonization and human exposure has been proposed. However, the highest prevalence of C. gattii cryptococcosis occurs in Papua New Guinea and in regions of Australia where the eucalypt species implicated to date are not endemic. This study investigated the population structure of three geographically distinct clinical and veterinary populations of C. gattii from Australia and Papua New Guinea. All populations that consisted of a genotype found frequently in Australia (VGI) were strongly clonal and were highly differentiated from one another. Two populations of the less common VGII genotype from Sydney and the Northern Territory had population structures inferring recombination. In addition, there was some evidence of reduced genetic differentiation between these geographically remote regions. In a companion study presented in this issue, VGII isolates were overwhelmingly more fertile than those of the VGI genotype, giving biological support to the indirect assessment of sexual exchange. It appears that the VGI genotype propagates clonally on eucalypts in Australia and on an unknown substrate in Papua New Guinea, with infection initiated by an unidentified infectious propagule. VGII isolates are completing their life cycles and may be dispersed via sexually produced basidiospores, which are also likely to initiate respiratory infection.

Clinical and Environmental Isolates of Cryptococcus gattii from Australia That Retain Sexual Fecundity

ABSTRACT Cryptococcus gattii is a primary pathogenic yeast that causes disease in both animals and humans. It is closely related to Cryptococcus neoformans and diverged from a common ancestor ∼40 million years ago. While C. gattii has a characterized sexual cycle dependent upon a dimorphic region of the genome known as the MAT locus, mating has rarely been observed in this species. In this study, we identify for the first time clinical (both human and veterinary) and environmental isolates from Australia that retain sexual fecundity. A collection of 120 isolates from a variety of geographic locations was analyzed for molecular type, mating type, and the ability to develop mating structures when cocultured with fertile tester strains. Nine isolates produced dikaryotic filaments with paired nuclei, fused clamp connections, and basidiospores. DNA sequence analysis of three genes (URA5, the MATα-specific SXI1α gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATα strains sequenced, the SXI1α gene was found to exist as 11 different alleles. Phylogenetic analysis found most variation to occur in the more fertile genotypes. Although some lineages of Australian C. gattii have retained the ability to mate, the majority of isolates were sterile, suggesting that asexuality is the dominant mode of propagation in these populations.

The effect of standard heat and filtration processing procedures on antimicrobial activity and hydrogen peroxide levels in honey

There is increasing interest in the antimicrobial properties of honey. In most honey types, antimicrobial activity is due to the generation of hydrogen peroxide (H2O2), but this can vary greatly among samples. Honey is a complex product and other components may modulate activity, which can be further affected by commercial processing procedures. In this study we examined honey derived from three native Australian floral sources that had previously been associated with H2O2-dependent activity. Antibacterial activity was seen in four red stringybark samples only, and ranged from 12 to 21.1% phenol equivalence against Staphylococcus aureus. Antifungal activity ranged from MIC values of 19–38.3% (w/v) against Candida albicans, and all samples were significantly more active than an osmotically equivalent sugar solution. All honey samples were provided unprocessed and following commercial processing. Processing was usually detrimental to antimicrobial activity, but occasionally the reverse was seen and activity increased. H2O2 levels varied from 0 to 1017 μM, and although samples with no H2O2 had little or no antimicrobial activity, some samples had relatively high H2O2 levels yet no antimicrobial activity. In samples where H2O2 was detected, the correlation with antibacterial activity was greater in the processed than in the unprocessed samples, suggesting other factors present in the honey influence this activity and are sensitive to heat treatment. Antifungal activity did not correlate with the level of H2O2 in honey samples, and overall it appeared that H2O2 alone was not sufficient to inhibit C. albicans. We conclude that floral source and H2O2 levels are not reliable predictors of the antimicrobial activity of honey, which currently can only be assessed by standardized antimicrobial testing. Heat processing should be reduced where possible, and honey destined for medicinal use should be retested post-processing to ensure that activity levels have not changed.

Global Transcriptome Profile of Cryptococcus neoformans during Exposure to Hydrogen Peroxide Induced Oxidative Stress

The ability of the opportunistic fungal pathogen Cryptococcus neoformans to resist oxidative stress is one of its most important virulence related traits. To cope with the deleterious effect of cellular damage caused by the oxidative burst inside the macrophages, C. neoformans has developed multilayered redundant molecular responses to neutralize the stress, to repair the damage and to eventually grow inside the hostile environment of the phagosome. We used microarray analysis of cells treated with hydrogen peroxide (H2O2) at multiple time points in a nutrient defined medium to identify a transcriptional signature associated with oxidative stress. We discovered that the composition of the medium in which fungal cells were grown and treated had a profound effect on their capacity to degrade exogenous H2O2. We determined the kinetics of H2O2 breakdown by growing yeast cells under different conditions and accordingly selected an appropriate media composition and range of time points for isolating RNA for hybridization. Microarray analysis revealed a robust transient transcriptional response and the intensity of the global response was consistent with the kinetics of H2O2 breakdown by treated cells. Gene ontology analysis of differentially expressed genes related to oxidation-reduction, metabolic process and protein catabolic processes identified potential roles of mitochondrial function and protein ubiquitination in oxidative stress resistance. Interestingly, the metabolic pathway adaptation of C. neoformans to H2O2 treatment was remarkably distinct from the response of other fungal organisms to oxidative stress. We also identified the induction of an antifungal drug resistance response upon the treatment of C. neoformans with H2O2. These results highlight the complexity of the oxidative stress response and offer possible new avenues for improving our understanding of mechanisms of oxidative stress resistance in C. neoformans.

Synergy and antagonism between iron chelators and antifungal drugs in Cryptococcus

Fungal infections remain very difficult to treat, and developing new antifungal drugs is difficult and expensive. Recent approaches therefore seek to augment existing antifungals with synergistic agents that can lower the therapeutic dose, increase efficacy and prevent resistance from developing. Iron limitation can inhibit microbial growth, and iron chelators have been employed to treat fungal infections. In this study, chequerboard testing was used to explore combinations of iron chelators with antifungal agents against pathogenic Cryptococcus spp. with the aim of determining how disruption to iron homeostasis affects antifungal susceptibility. The iron chelators ethylenediaminetetraacetic acid (EDTA), deferoxamine (DFO), deferiprone (DFP), deferasirox (DSX), ciclopirox olamine and lactoferrin (LF) were paired with the antifungal agents amphotericin B (AmB), fluconazole, itraconazole, voriconazole and caspofungin. All chelators except for DFO increased the efficacy of AmB, and significant synergy was seen between AmB and LF for all Cryptococcus strains. Addition of exogenous iron rescued cells from the antifungal effect of LF alone but could not prevent inhibition by AmB + LF, indicating that synergy was not due primarily to iron chelation but to other properties of LF that were potentiated in the presence of AmB. Significant synergy was not seen consistently for other antifungal-chelator combinations, and EDTA, DSX and DFP antagonised the activity of azole drugs in strains of Cryptococcus neoformans var. grubii. This study highlights the range of interactions that can be induced by chelators and indicates that most antifungal drugs are not enhanced by iron limitation in Cryptococcus.

Time-Course Proteome Analysis Reveals the Dynamic Response of Cryptococcus gattii Cells to Fluconazole

Cryptococcus gattii is an encapsulated fungus capable of causing fatal disease in immunocompetent humans and animals. As current antifungal therapies are few and limited in efficacy, and resistance is an emerging issue, the development of new treatment strategies is urgently required. The current study undertook a time-course analysis of the proteome of C. gattii during treatment with fluconazole (FLC), which is used widely in prophylactic and maintenance therapies. The aims were to analyze the overall cellular response to FLC, and to find fungal proteins involved in this response that might be useful targets in therapies that augment the antifungal activity of FLC. During FLC treatment, an increase in stress response, ATP synthesis and mitochondrial respiratory chain proteins, and a decrease in most ribosomal proteins was observed, suggesting that ATP-dependent efflux pumps had been initiated for survival and that the maintenance of ribosome synthesis was differentially expressed. Two proteins involved in fungal specific pathways were responsive to FLC. An integrative network analysis revealed co-ordinated processes involved in drug response, and highlighted hubs in the network representing essential proteins that are required for cell viability. This work demonstrates the dynamic cellular response of a typical susceptible isolate of C. gattii to FLC, and identified a number of proteins and pathways that could be targeted to augment the activity of FLC.

The Antibacterial Activity of Australian Leptospermum Honey Correlates with Methylglyoxal Levels

Most commercially available therapeutic honey is derived from flowering Leptospermum scoparium (manuka) plants from New Zealand. Australia has more than 80 Leptospermum species, and limited research to date has found at least some produce honey with high non-peroxide antibacterial activity (NPA) similar to New Zealand manuka, suggesting Australia may have a ready supply of medical-grade honey. The activity of manuka honey is largely due to the presence of methylglyoxal (MGO), which is produced non-enzymatically from dihydroxyacetone (DHA) present in manuka nectar. The aims of the current study were to chemically quantify the compounds contributing to antibacterial activity in a collection of Australian Leptospermum honeys, to assess the relationship between MGO and NPA in these samples, and to determine whether NPA changes during honey storage. Eighty different Leptospermum honey samples were analysed, and therapeutically useful NPA was seen in samples derived from species including L. liversidgei and L. polygalifolium. Exceptionally high levels of up to 1100 mg/kg MGO were present in L. polygalifolium honey samples sourced from the Northern Rivers region in NSW and Byfield, QLD, with considerable diversity among samples. There was a strong positive relationship between NPA and MGO concentration, and DHA was present in all of the active honey samples, indicating a potential for ongoing conversion to MGO. NPA was stable, with most samples showing little change following seven years of storage in the dark at 4°C. This study demonstrates the potential for Australian Leptospermum honey as a wound care product, and argues for an extension of this analysis to other Leptospermum species.

Cryptococcus Strains with Different Pathogenic Potentials Have Diverse Protein Secretomes

ABSTRACT Secreted proteins are the frontline between the host and pathogen. In mammalian hosts, secreted proteins enable invasive infection and can modulate the host immune response. Cryptococcosis, caused by pathogenic Cryptococcus species, begins when inhaled infectious propagules establish to produce pulmonary infection, which, if not resolved, can disseminate to the central nervous system to cause meningoencephalitis. Strains of Cryptococcus species differ in their capacity to cause disease, and the mechanisms underlying this are not well understood. To investigate the role of secreted proteins in disease, we determined the secretome for three genome strains of Cryptococcus species, including a hypovirulent and a hypervirulent strain of C. gattii and a virulent strain of C. neoformans. Sixty-seven unique proteins were identified, with different numbers and types of proteins secreted by each strain. The secretomes of the virulent strains were largely limited to proteolytic and hydrolytic enzymes, while the hypovirulent strain had a diverse secretome, including non-conventionally secreted canonical cytosolic and immunogenic proteins that have been implicated in virulence. The hypovirulent strain cannot establish pulmonary infection in a mouse model, but strains of this genotype have caused human meningitis. To directly test brain infection, we used intracranial inoculation and found that the hypovirulent strain was substantially more invasive than its hypervirulent counterpart. We suggest that immunogenic proteins secreted by this strain invoke a host response that limits pulmonary infection but that there can be invasive growth and damage if infection reaches the brain. Given their known role in virulence, it is possible that non-conventionally secreted proteins mediate this process.

Multilocus variable-number tandem-repeat analysis of clinical isolates of Aspergillus flavus from Iran reveals the first cases of Aspergillus minisclerotigenes associated with human infection.

BackgroundAspergillus flavus is intensively studied for its role in infecting crop plants and contaminating produce with aflatoxin, but its role as a human pathogen is less well understood. In parts of the Middle East and India, A. flavus surpasses A. fumigatus as a cause of invasive aspergillosis and is a significant cause of cutaneous, sinus, nasal and nail infections.MethodsA collection of 45 clinical and 10 environmental A. flavus isolates from Iran were analysed using Variable-Number Tandem-Repeat (VNTR) markers with MICROSAT and goeBURST to determine their genetic diversity and their relatedness to clinical and environmental A. flavus isolates from Australia. Phylogeny was assessed using partial β-tubulin and calmodulin gene sequencing, and mating type was determined by PCR. Antifungal susceptibility testing was performed on selected isolates using a reference microbroth dilution method.ResultsThere was considerable diversity in the A. flavus collection, with no segregation on goeBURST networks according to source or geographic location. Three Iranian isolates, two from sinus infections and one from a paranasal infection grouped with Aspergillus minisclerotigenes, and all produced B and G aflatoxin. Phylogenic analysis using partial β-tubulin and calmodulin sequencing confirmed two of these as A. minisclerotigenes, while the third could not be differentiated from A. flavus and related species within Aspergillus section flavi. Based on epidemiological cut-off values, the A. minisclerotigens and A. flavus isolates tested were susceptible to commonly used antifungal drugs.ConclusionsThis is the first report of human infection due to A. minisclerotigenes, and it raises the possiblity that other species within Aspergillus section flavi may also cause clinical disease. Clinical isolates of A. flavus from Iran are not distinct from Australian isolates, indicating local environmental, climatic or host features, rather than fungal features, govern the high incidence of A. flavus infection in this region. The results of this study have important implications for biological control strategies that aim to reduce aflatoxin by the introduction of non-toxigenic strains, as potentially any strain of A. flavus, and closely related species like A. minisclerotigenes, might be capable of human infection.

Species in the Cryptococcus gattii Complex Differ in Capsule and Cell Size following Growth under Capsule-Inducing Conditions.

Infections with the fungal pathogen Cryptococcus gattii have been increasing in recent years. Recently, four different species have been described within C. gattii, which correspond to four previously known molecular genotypes (VGI to VGIV). Examining traits related to infection and disease is important for determining whether these different species have clinical relevance. This study examined variation in attributes that are important for infecting and surviving in the host, including tolerance to various stresses, yeast cell size, and the amount of polysaccharide capsule that covers the cell. The cell size and capsule size were significantly different and inversely correlated across the species. Thermotolerance was highest in C. deuterogattii (VGII), the only species known to cause outbreaks, while most strains of the species C. bacillisporus (VGIII) and C. tetragattii (VGIV) grew poorly at 37°C. These findings argue for increased acceptance of the new species and may be useful for informing diagnosis and prognosis in clinical infection. ABSTRACT Cryptococcus gattii causes invasive fungal infections that have been increasing in incidence and global distribution in recent years. The major molecular genotypes of C. gattii that were previously classified as VGI to VGIV have recently been described as four new species: C. gattii (VGI), C. deuterogattii (VGII), C. bacillisporus (VGIII), and C. tetragattii (VGIV). The main driver for their classification has been phylogeny, and phenotypic diversity has not yet been extensively characterized. This study examines variation in attributes related to virulence and pathogenicity, including capsule thickness, cell size, tolerance to temperature, oxidative and osmotic stress, and cell wall integrity. A capsule induction agar using diluted Sabouraud medium revealed significant differences in capsule and cell size across the C. gattii species complex and produced irregularly shaped elongated cells in a number of strains. C. gattii/VGI strains possessed the largest capsules of all species but had smaller cells, while C. deuterogattii/VGII strains possessed the largest cells of all species but had smaller capsules. Overall thermotolerance was highest in C. deuterogattii/VGII strains, while a number of C. bacillisporus/VGIII, and C. tetragattii/VGIV strains had substantially reduced growth at 37°C. There was no significant difference among species in their tolerances to oxidative or osmotic stresses, and there was no evidence for defects in cell wall integrity in strains producing irregular cells. These data support the division of the C. gattii species complex into distinctly identified species and suggest underlying reasons for their differences in virulence, epidemiology, and host preference. IMPORTANCE Infections with the fungal pathogen Cryptococcus gattii have been increasing in recent years. Recently, four different species have been described within C. gattii, which correspond to four previously known molecular genotypes (VGI to VGIV). Examining traits related to infection and disease is important for determining whether these different species have clinical relevance. This study examined variation in attributes that are important for infecting and surviving in the host, including tolerance to various stresses, yeast cell size, and the amount of polysaccharide capsule that covers the cell. The cell size and capsule size were significantly different and inversely correlated across the species. Thermotolerance was highest in C. deuterogattii (VGII), the only species known to cause outbreaks, while most strains of the species C. bacillisporus (VGIII) and C. tetragattii (VGIV) grew poorly at 37°C. These findings argue for increased acceptance of the new species and may be useful for informing diagnosis and prognosis in clinical infection.