Spectrum of activity and mechanisms of azole-bisphosphonate synergy in pathogenic Candida.

Candidiasis places a significant burden on human health and can range from common superficial vulvovaginal and oral infections to invasive diseases with high mortality. The most common Candida species implicated in human disease is Candida albicans, but other species like Candida glabrata are emerging. The use of azole antifungals for treatment is limited by increasing rates of resistance. This study explores repositioning bisphosphonates, which are traditionally used for osteoporosis, as antifungal synergists that can improve and revitalize the use of azoles. Risedronate, alendronate, and zoledronate (ZOL) were tested against isolates from six different species of Candida, and ZOL produced moderate antifungal activity and strong synergy with azoles like fluconazole (FLC), particularly in C. glabrata. FLC:ZOL combinations had increased fungicidal and antibiofilm activity compared to either drug alone, and the combination prevented the development of antifungal resistance. Mechanistic investigations demonstrated that the synergy was mediated by the depletion of squalene, resulting in the inhibition of ergosterol biosynthesis and a compromised membrane structure. In C. glabrata, synergy compromised the function of membrane-bound multidrug transporters and caused an accumulation of reactive oxygen species, which may account for its acute sensitivity to FLC:ZOL. The efficacy of FLC:ZOL in vivo was confirmed in a Galleria mellonella infection model, where combinations improved the survival of larvae infected with C. albicans and C. glabrata to a greater extent than monotherapy with FLC or ZOL, and at reduced dosages. These findings demonstrate that bisphosphonates and azoles are a promising new combination therapy for the treatment of topical candidiasis. IMPORTANCE: Candida is a common and often very serious opportunistic fungal pathogen. Invasive candidiasis is a prevalent cause of nosocomial infections with a high mortality rate, and mucocutaneous infections significantly impact the quality of life of millions of patients a year. These infections pose substantial clinical challenges, particularly as the currently available antifungal treatment options are limited in efficacy and often toxic. Azoles are a mainstay of antifungal therapy and work by targeting the biosynthesis of ergosterol. However, there are rising rates of acquired azole resistance in various Candida species, and some species are considered intrinsically resistant to most azoles. Our research demonstrates the promising therapeutic potential of synergistically enhancing azoles with non-toxic, FDA-approved bisphosphonates. Repurposing bisphosphonates as antifungal synergists can bypass much of the drug development pipeline and accelerate the translation of azole-bisphosphonate combination therapy.

Increasing Lovastatin Production by Re-routing the Precursors Flow of Aspergillus terreus via Metabolic Engineering.

Lovastatin is an anti-cholesterol medicine that is commonly prescribed to manage cholesterol levels, and minimise the risk of suffering from heart-related diseases. Aspergillus terreus (ATCC 20542) supplied with carbohydrates or sugar alcohols can produce lovastatin. The present work explored the application of metabolic engineering in A. terreus to re-route the precursor flow towards the lovastatin biosynthetic pathway by simultaneously overexpressing the gene for acetyl-CoA carboxylase (acc) to increase the precursor flux, and eliminate ( +)-geodin biosynthesis (a competing secondary metabolite) by removing the gene for emodin anthrone polyketide synthase (gedC). Alterations to metabolic flux in the double mutant (gedCΔ*accox) strain and the effects of using two different substrate formulations were examined. The gedCΔ*accox strain, when cultivated with a mixture of glycerol and lactose, significantly (p < 0.05) increased the levels of metabolic precursors malonyl-CoA (48%) and acetyl-CoA (420%), completely inhibited the (+)-geodin biosynthesis, and increased the level of lovastatin [152 mg/L; 143% higher than the wild-type (WT) strain]. The present work demonstrated how the manipulation of A. terreus metabolic pathways could increase the efficiency of carbon flux towards lovastatin, thus elevating its overall production and enabling the use of glycerol as a substrate source. As such, the present work also provides a framework model for other medically or industrially important fungi to synthesise valuable compounds using sustainable carbon sources.

The Antifungal and Synergistic Effect of Bisphosphonates in Cryptococcus.

New treatment strategies are required for cryptococcosis, a leading mycosis in HIV-AIDS patients. Following the identification of Cryptococcus proteins differentially expressed in response to fluconazole, we targeted farnesyl pryrophosphate synthetase (FPPS), an enzyme in the squalene biosynthesis pathway, using nitrogenous bisphosphonates. We hypothesized that these would disrupt squalene synthesis and thereby produce synergy with fluconazole, which acts on a downstream pathway that requires squalene. The susceptibilities of 39 clinical isolates from 6 different species of Cryptococcus were assessed for bisphosphonates and fluconazole, used both independently and in combination. Effective fluconazole-bisphosphonate combinations were then assessed for fungicidal activity, efficacy against biofilms, and ability to resolve cryptococcosis in an invertebrate model. The nitrogenous bisphosphonates risedronate, alendronate, and zoledronate were antifungal against all strains tested. Zoledronate was the most effective (geometric mean MIC = 113.03 mg/liter; risedronate = 378.49 mg/liter; alendronate = 158.4 mg/liter) and was broadly synergistic when combined with fluconazole, with a fractional inhibitory concentration index (FICI) of ≤0.5 in 92% of isolates. Fluconazole and zoledronate in combination were fungicidal in a time-kill assay, inhibited Cryptococcus biofilms, prevented the development of fluconazole resistance, and resolved infection in a nematode model. Supplementation with squalene eliminated bisphosphonate-mediated synergy, demonstrating that synergy was due to the inhibition of squalene biosynthesis. This study demonstrates the utility of targeting squalene synthesis for improving the efficacy of azole-based antifungal drugs and suggests bisphosphonates are promising lead compounds for further antifungal development.

Different Pathways Mediate Amphotericin-Lactoferrin Drug Synergy in Cryptococcus and Saccharomyces.

Fungal infections are an increasing cause of morbidity and mortality. Current antifungal drugs are limited in spectrum, few new drugs are in development, and resistance is an increasing issue. Drug synergy can enhance available drugs and extend their lifetime, however, few synergistic combinations are in clinical use and mechanistic data on how combinations work is lacking. The multifunctional glycoprotein lactoferrin (LF) acts synergistically with amphotericin B (AMB) in a range of fungal species. Whole LF binds and sequesters iron, and LF can also be digested enzymatically to produce cationic peptides with distinct antimicrobial functions. To understand how LF synergizes AMB, we previously undertook a transcriptomic analysis in Saccharomyces and found a paradoxical down-regulation of iron and stress response, suggesting stress pathway interference was dysregulating an appropriate response, resulting in cell death. To extend this to a fungal pathogen, we here perform the same analysis in Cryptococcus neoformans. While both fungi responded to AMB in a similar way, the addition of LF produced remarkably contrasting results, with the Cryptococcus transcriptome enriched for processes relating to cellular stress, up-regulation of endoplasmic-reticulum-associated protein degradation (ERAD), stress granule disassembly and protein folding, endoplasmic reticulum-Golgi-vacuole trafficking and autophagy, suggesting an overall disruption of protein and lipid biosynthesis. These studies demonstrate that the mechanism of LF-mediated synergy is species-specific, possibly due to differences in the way LF peptides are generated, bind to and enter cells and act on intracellular targets, illustrating how very different cellular processes can underlie what appears to be a similar phenotypic response.

Improved lovastatin production by inhibiting (+)-geodin biosynthesis in Aspergillus terreus.

Lovastatin is widely prescribed to reduce elevated levels of cholesterol and prevent heart-related diseases. Cultivation of Aspergillus terreus (ATCC 20542) with carbohydrates or low-value feedstocks such as glycerol produces lovastatin as a secondary metabolite and (+)-geodin as a by-product. An A. terreus mutant strain was developed (gedCΔ) with a disrupted (+)-geodin biosynthesis pathway. The gedCΔ mutant was created by inserting the antibiotic marker hygromycin B (hyg) within the gedC gene that encodes emodin anthrone polyketide synthase (PKS), a primary gene responsible for initiating (+)-geodin biosynthesis. The effects of emodin anthrone PKS gene disruption on (+)-geodin and lovastatin biosynthesis and the production of the precursors acetyl-CoA and malonyl-CoA were investigated with cultures based on glycerol alone and in combination with lactose. The gedCΔ strain showed improved lovastatin production, particularly when cultivated on the glycerol-lactose mixture, increasing lovastatin production by 80% (113 mg/L) while simultaneously inhibiting (+)-geodin biosynthesis compared to the wild-type strain. This study thus shows that suppression of the (+)-geodin pathway increases lovastatin yield and demonstrates a practical approach of manipulating carbon flux by modulating enzyme activity.

Overexpression of acetyl-CoA carboxylase in Aspergillus terreus to increase lovastatin production.

The present work describes the application of homologous recombination techniques in a wild-type Aspergillus terreus (ATCC 20542) strain to increase the flow of precursors towards the lovastatin biosynthesis pathway. A new strain was generated to overexpress acetyl-CoA carboxylase (ACCase) by replacing the native ACCase promoter with a strong constitutive PadhA promoter from Aspergillus nidulans. Glycerol and a mixture of lactose and glycerol were used independently as the carbon feedstock to determine the degree of response by the A. terreus strains towards the production of acetyl-CoA, and malonyl-CoA. The new strain increased the levels of malonyl-CoA and acetyl-CoA by 240% and 14%, respectively, compared to the wild-type strain. As a result, lovastatin production was increased by 40% and (+)-geodin was decreased by 31% using the new strain. This study shows for the first time that the metabolism of Aspergillus terreus can be manipulated to attain higher levels of precursors and valuable secondary metabolites.

Transcriptome and network analyses in Saccharomyces cerevisiae reveal that amphotericin B and lactoferrin synergy disrupt metal homeostasis and stress response.

Invasive fungal infections are difficult to treat. The few available antifungal drugs have problems with toxicity or efficacy, and resistance is increasing. To overcome these challenges, existing therapies may be enhanced by synergistic combination with another agent. Previously, we found amphotericin B (AMB) and the iron chelator, lactoferrin (LF), were synergistic against a range of different fungal pathogens. This study investigates the mechanism of AMB-LF synergy, using RNA-seq and network analyses. AMB treatment resulted in increased expression of genes involved in iron homeostasis and ATP synthesis. Unexpectedly, AMB-LF treatment did not lead to increased expression of iron and zinc homeostasis genes. However, genes involved in adaptive response to zinc deficiency and oxidative stress had decreased expression. The clustering of co-expressed genes and network analysis revealed that many iron and zinc homeostasis genes are targets of transcription factors Aft1p and Zap1p. The aft1Δ and zap1Δ mutants were hypersensitive to AMB and H2O2, suggesting they are key regulators of the drug response. Mechanistically, AMB-LF synergy could involve AMB affecting the integrity of the cell wall and membrane, permitting LF to disrupt intracellular processes. We suggest that Zap1p- and Aft1p-binding molecules could be combined with existing antifungals to serve as synergistic treatments.

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.

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.

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

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.

Cryptococcus strains with different pathogenic potentials have diverse protein secretomes.

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.

BACKGROUND: Aspergillus 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. METHODS: A 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 ß-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. RESULTS: There 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 ß-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. CONCLUSIONS: This 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.

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 (H(2)O(2)) 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 H(2)O(2). We determined the kinetics of H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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 H(2)O(2). 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.

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 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 (H(2)O(2)), 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 H(2)O(2)-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. H(2)O(2) levels varied from 0 to 1017 µM, and although samples with no H(2)O(2) had little or no antimicrobial activity, some samples had relatively high H(2)O(2) levels yet no antimicrobial activity. In samples where H(2)O(2) 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 H(2)O(2) in honey samples, and overall it appeared that H(2)O(2) alone was not sufficient to inhibit C. albicans. We conclude that floral source and H(2)O(2) 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.

Cryptococcus neoformans, a fungus under stress.

Cryptococcus neoformans is a human fungal pathogen that survives exposure to stresses during growth in the human host, including oxidative and nitrosative stress, high temperature, hypoxia, and nutrient deprivation. There have been many genes implicated in resistance to individual stresses. Notably, the catalases do not have the expected role in resistance to external oxidative stress, but specific peroxidases appear to be critical for resistance to both oxidative and nitrosative stresses. Signal transduction through the HOG1 and calcineurin/calmodulin pathways has been implicated in the stress response. Microarray and proteomic analyses have indicated that the common responses to stress are induction of metabolic and oxidative stress genes, and repression of genes encoding translational machinery.

Looking for sex in the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii.

Why are we interested in understanding the mode of reproduction being used by the fungal pathogens Cryptococcus neoformans and Cryptococcus gattii? Empirical evidence has finally supported the long-held assumption that, by increasing the rate of adaptive evolution, sex increases the chances of long-term survival. Understanding the ability of pathogenic organisms to adapt to diagnostic and treatment regimes is also important in the fight against the diseases caused by these organisms. This review looks at the different approaches used to identify population structure in C. neoformans and C. gattii. These are sexual species; however, recombination in natural populations has only recently been found. We highlight the importance of population selection and the value of both indirect molecular analysis and direct biological evidence for sexual recombination, when looking for the mode of reproduction in these fungal pathogens.

Clonality and recombination in genetically differentiated subgroups of Cryptococcus gattii.

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.

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 approximately 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 MATalpha-specific SXI1alpha gene, and the MATa-specific SXI2a gene) revealed little or no variability in URA5 and SXI2a, respectively. However across the 108 MATalpha strains sequenced, the SXI1alpha 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.