Exposure of Cryptococcus neoformans to low nitrogen levels enhances virulence.

Previous studies have shown a correlation between nitrogen levels and Cryptococcus neoformans pathogenicity. Here we report on the in vivo effects of cryptococcal pre-exposure to ecologically relevant nitrogen levels. C. neoformans H99 was cultured in yeast carbon base (YCB) supplemented with 0.53 g/L NH4Cl and 0.21 g/L NH4Cl, respectively, and used to infect larvae of the Greater Wax moth, Galleria mellonella. Cells cultured in low nitrogen YCB (LN) were more virulent compared to cells cultured in high nitrogen YCB (HN). Microscopic examination of haemolymph collected from infected larvae revealed that cells cultured in LN were larger than cells cultured in HN, with the majority of LN cells exceeding 10 µm and possibly entering titanisation. Additionally, compared to HN-cultured cells, fewer LN-cultured cells were engulfed by macrophages. The enhanced virulence of LN-cultured cells was attributed to the increased cell size in vivo. In contrast, reduced macrophage uptake was attributed to increased capsule thickness of in vitro cells. Not only do these findings demonstrate the effects of culture conditions, specifically nitrogen levels, on C. neoformans virulence, but they also highlight the importance of isolate background in the cryptococcal-host interaction.

The pathogenesis of experimental Emergomycosis in mice.

Emergomyces africanus is a recently identified thermally-dimorphic fungal pathogen that causes disseminated infection in people living with advanced HIV disease. Known as emergomycosis, this disseminated disease is associated with very high case fatality rates. Over the last decade, improved diagnostics and fungal identification in South Africa resulted in a dramatic increase in the number of reported cases. Although the true burden of disease is still unknown, emergomycosis is among the most frequently diagnosed dimorphic fungal infections in Southern Africa; and additional species in the genus have been identified on four continents. Little is known about the pathogenesis and the host's immune response to this emerging pathogen. Therefore, we established a murine model of pulmonary infection using a clinical isolate, E. africanus (CBS 136260). Both conidia and yeast forms caused pulmonary and disseminated infection in mice with organisms isolated in culture from lung, spleen, liver, and kidney. Wild-type C57BL/6 mice demonstrated a drop in body weight at two weeks post-infection, corresponding to a peak in fungal burden in the lung, spleen, liver, and kidney. An increase in pro-inflammatory cytokine production was detected in homogenized lung supernatants including IFN-γ, IL-1ß, IL-6, IL12-p40 and IL-17 at three- and four-weeks post-infection. No significant differences in TNF, IL-12p70 and IL-10 were observed in wild-type mice between one and four-weeks post-infection. Rag-1-deficient mice, lacking mature T-and B-cells, had an increased fungal burden associated with reduced IFN-γ production. Together our data support a protective T-helper type-1 immune response to E. africanus infection. This may provide a possible explanation for the susceptibility of only a subset of people living with advanced HIV disease despite hypothesized widespread environmental exposure. In summary, we have established a novel murine model of E. africanus disease providing critical insights into the host immune components required for eliminating the infection.

Taxogenomic placement of Rasporella oleae and Rasporella dianae gen. and spp. nov., two insect associated yeast species.

During the course of independent studies in Europe, North America, and Africa, seven yeast strains were isolated from insect frass, decaying wood, tree flux, and olive oil sediment. Phylogenetic analysis of two barcoding DNA regions (internal transcribed spacer and the D1/D2 domain of the LSU rRNA gene) revealed that they belong to two closely related undescribed species distinct from all genera in the family Debaryomycetaceae. For reliable taxonomic placement the genomes of four strains of the two novel species and six type strains of closely related species were sequenced. Orthologous genes from 54 genomes of representatives of the Pichiomycetes and 23 outgroup taxa were concatenated to construct a fully supported phylogenetic tree. Consistent with the assumptions, we found that the two new species belong to a novel genus. In addition, the delimitation of the novel species was supported by genetic distance calculations from average nucleotide identity (ANI) and digital DNA:DNA hybridization (dDDH) values. The physiological characterization of the novel species was generally consistent with their genomic content. All strains had two alleles encoding secretory lipase in either two or three copies depending on the species. However, lipolytic activity was detected only in strains with three copies of the secretory lipase gene. Nevertheless, lipolytic activity might be related to their association with the insect gut. Based on these results, formal descriptions of the new genus Rasporella gen. nov. and of two new species Rasporella dianae sp. nov. (holotype UCDFST 68-643T , MycoBank no.: 850238) and Rasporella oleae sp. nov. (holotype ZIM 2471T , MycoBank no.: 850126) are provided.

Health risk posed by direct ingestion of yeasts from polluted river water.

River water is an essential human resource that may be contaminated with hazardous microorganisms. However, the risk of yeast infection through river water exposure is unclear because it is highly dependant on individual susceptibility and has therefore not been well-studied, to date. To evaluate this undefined risk, we analysed the fungal communities in less polluted (LP) and highly polluted (HP) river water, as determined using principal coordinate analysis of pollution indicators. We enumerated culturable yeasts using a thermally selective isolation procedure (37 °C) and thus promoted the growth of potentially opportunistic species. Yeast species identified as clinically relevant were then tested for antifungal resistance. In addition, we propose a quantitative microbial risk assessment (QMRA) framework to quantitatively assess the potential risk of yeast infection. Our results indicated that pollution levels significantly altered fungal communities (p = 0.007) and that genera representing opportunistic and pathogenic members were significantly more abundant in HP waters (p = 0.038). Additionally, the yeast species Candida glabrata and Clavispora lusitaniae positively correlated with other pollution indicators, demonstrating the species' indicator potential. Our QMRA results further indicate that higher risk of infection is associated with increased water pollution levels (considering both physicochemical and bacterial indicators). Furthermore, yeast species with higher pathogenic potential present an increased risk of infection despite lower observed concentrations in the river water. Interestingly, the bloom of Meyerozyma guilliermondii during the wet season suggests that other environmental factors, such as dissolved oxygen levels and water turbulence, might affect growth characteristics of yeasts in river water, which consequently affects the distribution of annual infection risks. The presence of antifungal resistant yeasts, observed in this study, could further contribute to variation in risk distribution. Research on the ecophysiology of yeasts in these environments is therefore necessary to ameliorate the uncertainty and sensitivity of the proposed QMRA model. In addition to the vital knowledge on opportunistic and pathogenic yeast occurrence in river water and their observed association with pollution, this study provides valuable methods and insights to initiate future QMRAs of yeast infections.

Physiological characterisation of Corynebacterium uterequi associated with pregnancy complications in mares.

Increasing cases of equine infertility and early embryonic loss in the Western Cape, South Africa, were documented in recent years. These appeared to be associated with Corynebacterium uterequi isolated from the uteri of infected mares. The purpose of this study was, therefore, to investigate the physiology and potential pathogenicity of this bacterium. Histopathological analyses were conducted on five mares suffering from reproductive complications, and from which Corynebacterium strains were detected on culture of uterine swabs. The histopathology revealed that the mares suffered from various forms of endometritis, suggesting a potential role of Corynebacterium strains in the disease. An isolate from one of the biopsies, and 11 other tentatively identified C. uterequi isolates from the urogenital tracts of other mares, which all had a history of pregnancy complications, were subsequently identified using molecular techniques and characterised based on environmental stress tolerance, enzyme profiles, antibiotic susceptibility and ability to form biofilms. It was found that representatives of C. uterequi possessed several virulence-associated characteristics, including trypsin and urease activity, as well as the ability to form weakly adherent monoculture biofilms. Several isolates displayed resistance to trimethoprim-sulfamethoxazole. In conclusion, this study provided some insight into the general physiology and pathogenic potential of C. uterequi, and points to the possible role of C. uterequi in the onset of equine pregnancy complications. Moreover, the ability to form biofilms suggests the potential for chronic infection, which was observed in 60% of the mares. Further research, however, is needed to implicate C. uterequi as an equine pathogen.

Predicting interactions of the frass-associated yeast Hyphopichia heimii with Olea europaea subsp. cuspidata and twig-boring bark beetles.

Bark beetles are destructive insect pests known to form symbioses with different fungal taxa, including yeasts. The aim of this study was to (1) determine the prevalence of the rare yeast Hyphopichia heimii in bark beetle frass from wild olive trees in South Africa and to (2) predict the potential interaction of this yeast with trees and bark beetles. Twenty-eight culturable yeast species were isolated from frass in 35 bark beetle galleries, including representatives of H. heimii from nine samples. Physiological characterization of H. heimii isolates revealed that none was able to degrade complex polymers present in hemicellulose; however, all were able to assimilate sucrose and cellobiose, sugars associated with an arboreal habitat. All isolates were able to produce the auxin indole acetic acid, indicative of a potential symbiosis with the tree. Sterol analysis revealed that the isolates possessed ergosterol quantities ranging from 3.644 ± 0.119 to 13.920 ± 1.230 mg/g dry cell weight, which suggested that H. heimii could serve as a source of sterols in bark beetle diets, as is known for other bark beetle-associated fungi. In addition, gas chromatography-mass spectrometry demonstrated that at least one of the isolates, Hyphopichia heimii CAB 1614, was able to convert the insect pheromone cis-verbenol to the anti-aggregation pheromone verbenone. This indicated that H. heimii could potentially influence beetle behaviour. These results support the contention of a tripartite symbiosis between H. heimii, olive trees, and bark beetles.

The copper transporter, Ctr4, and the microtubule-associated protein, Cgp1, are important for Cryptococcus neoformans adaptation to nitrogen availability.

Nitrogen limitation was previously shown to be an important regulator of several genes associated with virulence in Cryptococcus neoformans. Among the most highly expressed genes under low-nitrogen conditions were CTR4 and CGP1, encoding a copper transporter and a microtubule-associated protein, respectively. However, the functional association of these genes with nitrogen limitation-a nutritional stress experienced in both environment and host-remains to be determined. Moreover, whether increased CTR4 and CGP1 expression is linked to the enhanced cryptococcal drug tolerance previously observed in low-nitrogen conditions is yet to be elucidated. Therefore, the present study explored the role of Cgp1 and Ctr4 in C. neoformans nitrogen stress adaptation and antifungal susceptibility. Our results showed that these genes play a role in the growth of C. neoformans in nitrogen-limited media, nitrogen source assimilation and growth on nitrogen-poor woody debris. Furthermore, we demonstrate that both Ctr4 and Cgp1 contribute to oxidative stress and antifungal susceptibility, with a ctr4∆ mutant being more susceptible to fluconazole and a cgp1∆ mutant being more susceptible to fluconazole and amphotericin B. Overall, our findings improve our understanding of the role of Ctr4 and Cgp1 in cryptococcal drug tolerance and adaptation to nitrogen availability.

A link between urease and polyamine metabolism in Cryptococcus neoformans.

The urease enzyme of Cryptococcus neoformans is linked to different metabolic pathways within the yeast cell, several of which are involved in polyamine metabolism. Cryptococcal biogenic amine production is, however, largely unexplored and is yet to be investigated in relation to urease. The aim of this study was therefore to explore and compare polyamine metabolism in wild-type, urease-negative and urease-reconstituted strains of C. neoformans. Mass spectrometry analysis showed that agmatine and spermidine were the major extra- and intracellular polyamines of C. neoformans and significant differences were observed between 26 and 37 °C. In addition, compared to the wild-type, the relative percentages of extracellular putrescine and spermidine were found to be lower and agmatine higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. Cyclohexylamine was a more potent polyamine inhibitor compared to DL-α-difluoromethylornithine and inhibitory effects were more pronounced at 37 °C than at 26 °C. At both temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine treatment compared to the wild-type. For both inhibitors, growth inhibition was alleviated with polyamine supplementation. This study has provided novel insight into the polyamine metabolism of C. neoformans, highlighting the involvement of urease in biogenic amine production.

Transcriptomic response of Cryptococcus neoformans to ecologically relevant nitrogen concentrations.

Nitrogen availability is vital for the growth and survival of Cryptococcus neoformans in the natural environment. Two major ecological reservoirs were previously described for C. neoformans, namely, pigeon guano and the woody debris of various tree species. In contrast to the abundance of available nitrogen in guano, C. neoformans must adapt to severely limited nitrogen conditions within arboreal ecological niches. Previously, we demonstrated the role of nitrogen limitation in the production of cryptococcal virulence factors and drug tolerance. The genetic response underlying this adaptation to nitrogen deficiency, however, remains to be determined. Therefore, in the present study we investigated the transcriptomic response of C. neoformans to ecologically relevant nitrogen concentrations using RNA-sequencing. Our data revealed that low nitrogen conditions modulate the expression of numerous virulence genes in C. neoformans. Among these were, CTR4 and CGP1, which showed highly significant modulation under low nitrogen conditions. Furthermore, data analysis revealed the upregulation of antifungal tolerance-related genes in low nitrogen conditions, including genes involved in ergosterol biosynthetic processes and cell wall integrity. Overall, our findings provide insight into the survival of C. neoformans in nitrogen-poor ecological niches and suggest that pre-adaptation to these conditions may influence the pathobiology of this yeast.

The virulence factor urease and its unexplored role in the metabolism of Cryptococcus neoformans.

Cryptococcal urease is believed to be important for the degradation of exogenous urea that the yeast encounters both in its natural environment and within the human host. Endogenous urea produced by the yeast's own metabolic reactions, however, may also serve as a substrate for the urease enzyme. Using wild-type, urease-deletion mutant and urease-reconstituted strains of Cryptococcus neoformans H99, we studied reactions located up- and downstream from endogenous urea. We demonstrated that urease is important for cryptococcal growth and that, compared to nutrient-rich conditions at 26°C, urease activity is higher under nutrient-limited conditions at 37°C. Compared to cells with a functional urease enzyme, urease-deficient cells had significantly higher intracellular urea levels and also showed more arginase activity, which may act as a potential source of endogenous urea. Metabolic reactions linked to arginase were also affected, since urease-positive and urease-negative cells differed with respect to agmatinase activity, polyamine synthesis, and intracellular levels of proline and reactive oxygen species. Lastly, urease-deficient cells showed higher melanin levels at 26°C than wild-type cells, while the inverse was observed at 37°C. These results suggest that cryptococcal urease is associated with the functioning of key metabolic pathways within the yeast cell.

Nitrogen concentration affects amphotericin B and fluconazole tolerance of pathogenic cryptococci.

Environmental stress often causes phenotypic changes among pathogenic cryptococci, such as altered antifungal susceptibility, changes in capsule and melanin formation, as well as altered levels of the membrane sterol and antifungal target, ergosterol. We therefore hypothesised that nitrogen limitation, a prevalent environmental stress in the natural habitat of these yeasts, might affect virulence and antifungal susceptibility. We tested the effect of different nitrogen concentrations on capsule, melanin and ergosterol biosynthesis, as well as amphotericin B (AmB) and fluconazole (FLU) susceptibility. This was achieved by culturing cryptococcal strains representing Cryptococcus neoformans and Cryptococcus gattii in media with high (0.53 g/l), control (0.42 g/l) and low (0.21 g/l) NH4Cl concentrations. India ink staining was used to determine capsule thickness microscopically, while melanin and ergosterol content were determined spectrophotometrically. We found that lower nitrogen concentrations enhanced both ergosterol and capsule biosynthesis, while a variable effect was observed on melanisation. Evaluation of drug tolerance using time-kill methodology, as well as tests for FLU heteroresistance, revealed that the low nitrogen cultures had the highest survival percentages in the presence of both AmB and FLU, and showed the highest frequency of FLU heteroresistance, suggesting that nitrogen concentration may indeed influence drug tolerance.

Designed Antimicrobial Peptides for Recurrent Vulvovaginal Candidiasis Treatment.

Recurrent vulvovaginal candidiasis (RVVC) is a widespread chronic infection that has a substantial negative impact on work and quality of life. The development of antimicrobial resistance and biofilm formation are speculated to contribute to Candida pathogenicity and treatment ineffectiveness. Designed antimicrobial peptides (dAMPs) are chemically modified from endogenous antimicrobial peptides that provide the first line of defense against pathogens. The goal here is to identify a dAMP for the topical treatment of RVVC. The dAMP MICs were determined for 46 fluconazole-susceptible and fluconazole-resistant Candida spp. clinical isolates. The possibility of inducing dAMP drug resistance and comparison of dAMP and fluconazole activity against preformed Candida biofilm and biofilm formation were evaluated. Assessment of mammalian cell viability was determined using bioluminescent human keratinocytes. The dAMP effect on fungus was probed via scanning electron microscopy, and topically applied dAMP activity was evaluated in a rodent vulvovaginal candidiasis (VVC) infection model. dAMPs demonstrated broad-spectrum antimicrobial activity against common causative clinical Candida isolates, reduced preformed biofilm, and inhibited biofilm formation. An evaluated dAMP did not induce resistance after repeated exposure of Candida tropicalis The dAMPs were selective for Candida cells with limited mammalian cytotoxicity with substantial activity in a rodent VVC model. dAMPs are described as having potent antifungal and antibiofilm activity, likely direct membrane action with selectivity for Candida cells, with limited resistance development. Combined with activity in a rodent VVC model, the data support clinical evaluation of dAMPs for topical treatment of VCC and recurrent VVC infections.

Binary interactions between the yeast Candida albicans and two gut-associated Bacteroides species.

The yeast Candida albicans forms part of the natural gut microbiota of healthy human individuals and its interactions with other microbial symbionts can impact host well-being. We therefore studied binary interactions between potentially pathogenic representatives of the gut-associated bacterial genus Bacteroides and C. albicans using anaerobic bacteria/yeast co-cultures prepared with a quarter-strength brain heart infusion (» BHI; 9.25 g/l) broth. We found that, except for minor changes observed in the cell numbers of one out of four C. albicans strains tested, yeast growth was largely unaffected by the presence of the bacteria. In contrast, growth of Bacteroides fragilis NCTC 9343 and Bacteroides vulgatus ATCC 8482 was significantly enhanced in the presence of C. albicans. Supplementation of Bacteroides monocultures with dead Candida albicans CAB 392 cells, containing intact outer cell wall mannan layers, resulted in increased bacterial concentrations. Subsequent culturing of the Bacteroides strains in a liquid minimal medium supplemented with candidal mannan demonstrated that B. vulgatus ATCC 8482, unlike B. fragilis NCTC 9343, utilized the mannan. Furthermore, by reducing the initial oxygen levels in monocultures prepared with » BHI broth, bacterial numbers were significantly enhanced compared to in monocultures prepared with » BHI broth not supplemented with the reducing agent l-cysteine hydrochloride. This suggests that C. albicans can stimulate Bacteroides growth via aerobic respiration and/or antioxidant production. The cell-free supernatant of 24-h-old C. albicans CAB 392 monocultures was also found to increase Bacteroides growth and chloramphenicol sensitivity.

Draft Genome Sequence of the Lignocellulose-Degrading Ascomycete Coniochaeta p ulveracea CAB 683.

Coniochaeta pulveracea is a soft-rot-causing ascomycete able to degrade lignocellulosic biomass. The first draft genome sequence of strain CAB 683 reported here has an estimated size of 30 Mb assembled into 852 scaffolds and 10,035 predicted protein-coding genes.

Emergomyces africanus in Soil, South Africa.

We detected Emergomyces africanus, a thermally dimorphic fungus that causes an HIV-associated systemic mycosis, by PCR in 18 (30%) of 60 soil samples from a wide range of habitats in South Africa. Direct and indirect culture techniques were unsuccessful. Experimental intraperitoneal inoculation of conidia induced murine disease.

Molecular detection of airborne Emergomyces africanus, a thermally dimorphic fungal pathogen, in Cape Town, South Africa.

Emergomyces africanus is a thermally dimorphic fungus that causes a systemic mycosis in immunocompromised persons in South Africa. Infection is presumed to follow inhalation of airborne propagules. We developed a quantitative PCR protocol able to detect as few as 5 Es. africanus propagules per day. Samples were collected in Cape Town, South Africa over 50 weeks by a Burkard spore trap with an alternate orifice. We detected Es. africanus in air samples from 34 days (10%) distributed over 11 weeks. These results suggest environmental exposure to airborne Es. africanus propagules occurs more commonly in endemic areas than previously appreciated.

First report of urease activity in the novel systemic fungal pathogen Emergomyces africanus: a comparison with the neurotrope Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic pathogen responsible for the AIDS-defining illness, cryptococcal meningitis. During the disease process, entry of cryptococcal cells into the brain is facilitated by virulence factors that include urease enzyme activity. A novel species of an Emmonsia-like fungus, recently named Emergomyces africanus, was identified as a cause of disseminated mycosis in HIV-infected persons in South Africa. However, in contrast to C. neoformans, the enzymes produced by this fungus, some of which may be involved in pathogenesis, have not been described. Using a clinical isolate of C. neoformans as a reference, the study aim was to confirm, characterise and quantify urease activity in E. africanus clinical isolates. Urease activity was tested using Christensen's urea agar, after which the presence of a urease gene in the genome of E. africanus was confirmed using gene sequence analysis. Subsequent evaluation of colorimetric enzyme assay data, using Michaelis-Menten enzyme kinetics, revealed similarities between the substrate affinity of the urease enzyme produced by E. africanus (Km ca. 26.0 mM) and that of C. neoformans (Km ca. 20.6 mM). However, the addition of 2.5 g/l urea to the culture medium stimulated urease activity of E. africanus, whereas nutrient limitation notably increased cryptococcal urease activity.

Binary Interactions of Antagonistic Bacteria with Candida albicans Under Aerobic and Anaerobic Conditions.

We used both aerobic and anaerobic liquid co-cultures, prepared with Luria Bertani broth, to study the effect of bacteria on the survival of Candida albicans in the external environment, away from an animal host. The bacteria were represented by Aeromonas hydrophila, Bacillus cereus, Bacillus subtilis, Clostridium, Enterobacter, Klebsiella pneumoniae, Kluyvera ascorbata and Serratia marcescens. Under aerobic conditions, the yeast's growth was inhibited in the presence of bacterial growth; however, under anaerobic conditions, yeast and bacterial growth in co-cultures was similar to that observed for pure cultures. Subsequent assays revealed that the majority of bacterial strains aerobically produced extracellular hydrolytic enzymes capable of yeast cell wall hydrolysis, including chitinases and mannan-degrading enzymes. In contrast, except for the A. hydrophila strain, these enzymes were not detected in anaerobic bacterial cultures, nor was the antimicrobial compound prodigiosin found in anaerobic cultures of S. marcescens. When we suspended C. albicans cells in crude extracellular enzyme preparations from K. pneumoniae and S. marcescens, we detected no negative effect on yeast viability. However, we found that these preparations enhance the toxicity of prodigiosin towards the yeast, especially in combination with mannan-degrading enzymes. Analyses of the chitin and mannan content of yeast cell walls revealed that less chitin was produced under anaerobic than aerobic conditions; however, the levels of mannan, known for its low permeability, remained the same. The latter phenomenon, as well as reduced production of the bacterial enzymes and prodigiosin, may contribute to anaerobic growth and survival of C. albicans in the presence of bacteria.

The microcyclic conidial stage of Coniochaeta pulveracea and its effect on selected biological interactions.

Coniochaeta pulveracea is a dimorphic lignicolous fungus that has mostly been isolated from decaying wood. However, relatively little work was conducted on the conditions for the dimorphic switch or the biological interactions of the fungus in its yeast-like microcyclic growth phase. Therefore, in this study, the microcyclic conidiation of C. pulveracea strains and representatives of the closely related species, Coniochaeta boothii and Coniochaeta subcorticalis, was studied under different environmental conditions. The strains were found to exhibit hyphal growth on solid substrates and underwent a dimorphic switch to produce microcycle conidiation upon transfer to a liquid medium which differed in physico-chemical composition compared to the original solid medium. Factors that were found to contribute to this dimorphic switch were temperature, pH and the presence of complex nitrogen sources such as casamino acids and peptone in the medium. However, C. pulveracea showed intraspecific differences with regard to its response to changes in the physico-chemical environment. The interactions of microcyclic Coniochaeta strains with selected yeasts, such as representatives of Meyerozyma guilliermondii and Cryptococcus neoformans, were subsequently studied in complex liquid media and it was found that, depending on medium composition, the microcyclic Coniochaeta exerted different effects on the different yeasts strains. In some co-cultures, a positive effect on yeast growth was observed, whilst in other cases microcyclic Coniochaeta inhibited yeast growth.