Diversity of laccase among Cryptococcus neoformans serotypes

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.     

Sexual compatibility between serotypes ofFilobasidiella neoformans (Cryptococcus neoformans)

Twenty-five of 37Cryptococcus neoformans strains of known serotype produced the basidiomycetousFilobasidiella state either alone or when paired with a strain of compatible mating-type. Sixteen strains were mating-type, four strains were a mating-type, and five strains were self-fertile.F. neoformans serotypes A and D were interfertile with compatible mating-types ofF. bacillispora serotypes B and C.C. neoformans var.gattii was interfertile with compatible mating-types ofF. neoformans andF. bacillispora. F. bacillispora strains, which utilized creatinine andl-malic acid, were interfertile with compatible mating-types ofF. neoformans, which did not utilize creatinine andl-malic acid. The interfertility between serotypes and biotypes eliminates the need for recognizing the names ofC. neoformans var.gattii, C. bacillisporus, andF. bacillispora. It is proposed thatC. neoformans var.gattii andC. bacillisporus be regarded as later, facultative synonyms ofC. neoformans and thatF. bacillispora be regarded as a later, facultative synonym ofF. neoformans.     

Biochemical differences between serotypes of Cryptococcus neoformans.

Prior studies have shown that C. neoformans isolates belonging to serotypes B and C differed from serotype A and D isolates in respect to the morphology of the perfect state, geographic distribution within the U.S.A. and frequency of isolation from avian droppings. The current study found that uptake of radiolabeled l-malic acid was approximately tenfold greater in serotype B and C than in A and D isolates. Assimilation of l-malic, fumaric and succinic acids also distinguished serotypes B and C from A and D. Greening of Guizotia seed agar occurred with 18 of 32 serotype B and C but in none of 91 serotype A or D isolates. The one property not following the same line of division was relatively slow creatinine assimilation, which distinguished type A alone.     

Varietes and serotypes of Cryptococcus neoformans clinical isolates in Colombia

The aim of this study was to contribute to the knowledge of the geographic distribution of Cryptococcus neoformans varieties, in Colombian patients, and to determine the mating types of such varieties. A total of 370 clinical isolates were studied. C. neoformans var. neoformans was identified in 95.2% of them. C. neoformans var. gattii in 4.8%, 4.5% being serotype B and 0.3%, serotype C. Fifty-five of the 74 (74%) isolates studied were mating type "alpha", all of them C. neoformans var. neoformans. Serotype C had been previously reported only once in South American countries.     

Mating types and serotypes of Cryptococcus neoformans isolated in Japan

Thirty-two isolates of Cryptococcus neoformans from patients and one from the wild obtained in Japan were characterized for their serotype, self-fertility, and mating behaviour by crossing them with two mating types of Filobasidiella neoformans var. neoformans and F. neoformans var. bacillispora. Of the 32 isolates from patients, 31 were of serotype A and the remaining one was of serotype D. Although these 32 isolates were all self-sterile, 23 serotype A and one serotype D isolates produced a complete sexual state when mixed with the alpha mating type of F. neoformans var. neoformans. The one natural isolate was of serotype A-D and self-fertile. The Japanese clinical isolates of C. neoformans appear to be predominantly serotype A and alpha mating type of F. neoformans var. neoformans as is the case in the U.S.A.     

In vitro antifungal susceptibility of clinical isolates of Cryptococcus neoformans var. neoformans and C. neoformans var. gattii

One of the differences observed between the two varieties of Cryptococcus neoformansis the greater difficulty to achieve an adequate therapeutical response in patients affected by C. neoformans var. gattii, an observation that has been validated in vitro only rarely. The aim of this work was to study the susceptibility patterns of 35 Colombian clinical isolates of C. neoformans, 20 of which belonged to the var. neoformans and 15 to the var. gattii. The minimal inhibitory concentration (MIC) was determined by broth microdilution, according to a modification of the methodology proposed by the National Committee for Clinical Laboratory Standards (NCCLS), using the breakpoints recently suggested by Nguyen et al. (Antimicrob Agents Chemother 1998; 42: 471-472). The antifungals tested were amphotericin B, fluconazole and itraconazole. Most of the isolates were susceptible to the three antimycotics tested regardless of the variety. Resistance to amphotericin B (MIC=2 microg/ml) was documented in two (10%) C. neoformans var. neoformans isolates; additionally, five (33%) C. neoformans var. gattii isolates felt in the category of fluconazole susceptible but dose dependent (MIC 16 microg/ml). In general, all C. neoformans var. gattii isolates proved susceptible only to the higher concentrations of the antifungals tested. For amphotericin B, seven (47%) isolates of this variety had MICs of 1 microg/ml, for fluconazole there were seven (47%) with MICs of 8 microg/ml and in the case of itraconazole, 10 isolates (66%) had MICs > 0.03 microg/ml. The data showed that although these isolates would be classified as susceptible, they actually require greater concentrations of the antifungals to be inhibited. This finding may well correlate both with the difficulty to attain therapeutic success in patients affected with C. neoformans var. gattii and with the need for more prolonged treatment courses in such cases.     

Combatting the evolution of antifungal resistance in Cryptococcus neoformans

Fungal infections are a global concern and the evolution of intrinsic resistance to current antifungals presents an alarming problem. For Cryptococcus neoformans, a human fungal pathogen of primarily immunocompromised individuals, resistance toward treatment strategies demands alternative approaches. Given the prevalence of virulence factor production during cryptococcal infection, an emerging and important field of research encompasses the development of novel antivirulence therapies proposed to improve host immune responses and promote fungal clearance. To accomplish this task, information regarding the presence and role of virulence factors, the mechanisms of action within the host, and the ability to influence fungal susceptibility to antifungals is pertinent. Research into mechanisms of antifungal resistance for C. neoformans is limited but extrapolation from successful studies in other fungal species can improve our understanding of mechanisms employed by C. neoformans and suggest targeted strategies to enhance our ability to combat the pathogen. In this Review, we highlight antifungal therapy options against Cryptococcus, explore current knowledge of underlying mechanisms promoting resistance, and present new opportunities for novel and effective strategies to overcome fungal infections and reduce, or possibly even reverse, the effects of resistance evolution.     

Synthesis of new antifungal peptides selective against Cryptococcus neoformans

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore there remains an urgent need for a new generation of antifungal agents. According to a polypharmacological approach, the present work concerns the synthesis and antifungal activity of a set of peptides designed to simultaneously target the fungal cell surface and lanosterol demethylase, a key enzyme involved in ergosterol synthesis. Our peptides include amino acid sequences characteristic of membrane-active antimicrobial peptides (AMP), and due to the presence of His residues, they carry the imidazole ring characteristic of azole compounds. The peptides synthesized by us, were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans.     

Many globally isolated AD hybrid strains of Cryptococcus neoformans originated in Africa

Interspecific and intervarietal hybridization may contribute to the biological diversity of fungal populations. Cryptococcus neoformans is a pathogenic yeast and the most common fungal cause of meningitis in patients with AIDS. Most patients are infected with either of the two varieties of C. neoformans, designated as serotype A (C. neoformans var. grubii) or serotype D (C. neoformans var. neoformans). In addition, serotype AD strains, which are hybrids of these two varieties, are commonly isolated from clinical and environmental samples. While most isolates of serotype A and serotype D are haploid, AD strains are diploid or aneuploid, and contain two sets of chromosomes and two mating type alleles, MATa and MATalpha, one from each of the serotypes. The global population of serotype A is dominated by isolates with the MATalpha mating type (Aalpha); however, about half of the globally analyzed AD strains possess the extremely rare serotype A MATa allele (Aa). We previously described an unusual population of serotype A in Botswana, in which 25% of the strains contain the rare MATa allele. Here we utilized two methods, phylogenetic analysis of three genes and genotyping by scoring amplified fragment length polymorphisms, and discovered that AD hybrid strains possessing the rare serotype A MATa allele (genotype AaDalpha) cluster with isolates of serotype A from Botswana, whereas AD hybrids that possess the MATalpha serotype A allele (AalphaDa and AalphaDalpha) cluster with cosmopolitan isolates of serotype A. We also determined that AD hybrid strains are more resistant to UV irradiation than haploid serotype A strains from Botswana. These findings support two hypotheses: (i) AaDalpha strains originated in sub-Saharan Africa from a cross between strains of serotypes A and D; and (ii) this fusion produced hybrid strains with increased fitness, enabling the Botswanan serotype A MATa genome, which is otherwise geographically restricted, to survive, emigrate, and propagate throughout the world.     

alpha AD alpha hybrids of Cryptococcus neoformans: evidence of same-sex mating in nature and hybrid fitness

Cryptococcus neoformans is a ubiquitous human fungal pathogen that causes meningoencephalitis in predominantly immunocompromised hosts. The fungus is typically haploid, and sexual reproduction involves two individuals with opposite mating types/sexes, alpha and a. However, the overwhelming predominance of mating type (MAT) alpha over a in C. neoformans populations limits alpha-a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between alpha isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural alpha AD alpha hybrids that arose by fusion between two alpha cells of different serotypes (A and D) were identified and characterized, providing definitive evidence that same-sex mating occurs naturally. A novel truncated allele of the mating-type-specific cell identity determinant SXI1 alpha was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed alpha AD alpha strains exhibited hybrid vigor both in vitro and in vivo, providing a plausible explanation for their relative abundance in nature despite the fact that AD hybrids are inefficient in meiosis/sporulation and are trapped in the diploid state. These findings provide insights on the origins, genetic mechanisms, and fitness impact of unisexual hybridization in the Cryptococcus population.     

Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target

Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, gamma-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.     

Urease activity in Cryptococcus neoformans and Cryptococcus gattii

Urease is an enzyme considered one of the main virulence factors in Cryptococcus neoformans. Quantitative differences in urease production between C. neoformans and the new species Cryptococcus gattii have not been so far documented. Using a standardized method, 25 isolates of C. neoformans and 19 of C. gattii were seeded in Christensen urea broth medium for urease activity detection. Approximately, the 50% of activity of one unit of commercial jack beans urease (A550=0.215) was considered as a reference to classified the Cryptococcus in two cathegories, low (A550<0.215) or high (A550=or>0.215) urease producers. After 72 hours of incubation, 76% of C. neoformans and 15.8% of C. gattii strains were high urease producers (p=0.016). Based on these results, the species C. neoformans appeared as the highest urease producer. Other virulence factors should also be investigated to explain C. gattii pathogenicity.     

Melanogenesis in Cryptococcus neoformans

Melanogenesis in Cryptococcus neoformans begins with the oxidation of dihydroxyphenylalanine by the enzyme phenol oxidase. The succeeding steps are very rapid. Two intermediates, dopachrome and 5,6-dihydroxyindole, have been isolated and characterized by high performance liquid chromatography. A pathway of melanin formation in C. neoformans is proposed, based on the presence of these intermediates.     

舍曲林抗新生隐球菌的体外及动物实验研究

目的评估舍曲林抗新生隐球菌的效果。方法实验分为6组,分别为空白对照组、10 mg/mL氟康唑、10 mg/mL舍曲林、20 mg/mL舍曲林、10 mg/mL氟康唑联用10 mg/mL舍曲林以及10 mg/mL氟康唑联用20 mg/mL舍曲林组。通过体外药敏试验及BALBc小鼠新生隐球菌动物探讨各组间抗隐球菌效果的差异。结果体外药敏试验发现舍曲林可有效降低新生隐球菌菌落数,当与氟康唑联用时抑菌效果更显著。动物实验发现2种浓度的舍曲林都可明显降低感染小鼠实验早期脑、肺组织的新生隐球菌菌落数,但在实验后期,低浓度的舍曲林对感染小鼠脑组织失去抑菌作用。脑、肺组织中,舍曲林治疗对新生隐球菌的抗菌效果均不如氟康唑。舍曲林与氟康唑联合用药对新生隐球菌模型小鼠肺组织的抗菌效果强于单用舍曲林或氟康唑。结论舍曲林具有抗新生隐球菌的作用,当与氟康唑联合用药时可起到协同作用。     

Virulence and antifungal susceptibility of environmental and clinical isolates of Cryptococcus neoformans from Puerto Rico

A case of cerebral cladosporiosis caused by Cladosporium trichoides (bantianum) now known as Xylohypha bantiana is described and illustrated. Predisposing debilitating diseases were not detectable. The Cladosporiosis diagnosis was based on visualisation of hyphal element in direct Gram's stain, direct KOH preparate of pus from brain abscess and on repeated successful cultivation of Cladosporium trichoides from specimen and by histopathology. Following surgery and anti-fungal chemotherapy the patient was cured.     

Scaffold hopping of sampangine: Discovery of potent antifungal lead compound against Aspergillus fumigatus and Cryptococcus neoformans

Discovery of novel antifungal agents against Aspergillus fumigatus and Cryptococcus neoformans remains a significant challenge in current antifungal therapy. Herein the antifungal natural product sampangine was used as the lead compound for novel antifungal drug discovery. A series of D-ring scaffold hopping derivatives were designed and synthesized to improve antifungal activity and water solubility. Among them, the thiophene derivative S2 showed broad-spectrum antifungal activity, particularly for Aspergillus fumigatus and Cryptococcus neoformans. Moreover, compound S2 also revealed better water solubility than sampangine, which represents a promising antifungal lead compound for further structural optimization.     

Creatinine metabolism in Cryptococcus neoformans and Cryptococcus bacillisporus.

The pathogenic species of Cryptococcus, C. neoformans and C. bacillisporus, utilized creatinine as a source of nitrogen but not of carbon. Chromatographic and autoradiographic studies suggest that creatinine metabolism in both species involves a single step resulting in the production of methylhydantoin and ammonia. The enzyme responsible for this step, creatinine deiminase, was produced by the cells only in the presence of creatinine in both species. The synthesis of creatinine deiminase was repressed by ammonia in C. neoformans, but not in C. bacillisporus. A possible explanation for this variation, based on the ecological differences between the two species, is discussed. A novel method for measuring creatinine deiminase activity is also described.