αADα 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, α and a. However, the overwhelming predominance of mating type (MAT) α over a in C. neoformans populations limits α–a mating in nature. Recently it was discovered that C. neoformans can undergo same-sex mating under laboratory conditions, especially between α isolates. Whether same-sex mating occurs in nature and contributes to the current population structure was unknown. In this study, natural αADα hybrids that arose by fusion between two α 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 SXI1α was also identified as a genetic factor likely involved in this process. In addition, laboratory-constructed αADα 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.     

白地霉Cryytococcus neoformans脂肪酶的双水相萃取

本文研究了不同无机盐的双水相体系对白地霉脂肪酶的萃取分离效果,对PEG/（NH4）2SO4成相系统进行了系统的研究,通过考察体系PEG分子量、不同的无机盐、PEG浓度、（NH4）2SO4浓度、离子强度、pH值及（NH4）2SO4浓度对反萃取的影响,并通过正交实验进一步优化了实验条件,初步确定在PEG浓度15%,（NH4）2SO4浓度22.5%,pH8.0,不加NaCl的条件下进行双水相萃取,脂肪酶分离系数和纯化倍数分别为6.8和7.5,比活力达到40.3 U/mg蛋白。     

Molecular diversity of serial Cryptococcus neoformans isolates from AIDS patients in the city of São Paulo, Brazil

Despite highly active anti-retroviral therapy, cryptococcal meningoencephalitis is the second most prevalent neurological disease in Brazilian AIDS patients, being frequently a defining condition with several episodes. As knowledge of Cryptococcus neoformans isolates in the same episode is critical for understanding why some patients develop several episodes, we investigated the genotype characteristics of C. neoformans isolates in two different situations. By pulsed field gel electrophoresis and random amplified polymorphic DNA analysis, 54 isolates from 12 patients with AIDS and cryptococcosis were analyzed. Group 1 comprised 39 isolates from nine patients with a single episode and hospitalization. Group 2 comprised 15 isolates from three patients with two episodes and hospitalizations. Except for three patients from group 1 probably infected with a single C. neoformans isolate, the other nine patients probably were infected with multiple isolates selected in different collection periods, or the infecting isolate might have underwent mutation to adapt and survive the host immune system and/or the antifungal therapy. However, the three patients from group 2 presented genetic diversity among isolates collected in both hospitalizations, possibly having hosted the initial isolate in both periods. These data, emphasize that Cryptococcus diversity in infection can contribute to strategies of treatment and prevention of cryptococcosis.     

Peroxisomal and Mitochondrial β-Oxidation Pathways Influence the Virulence of the Pathogenic Fungus Cryptococcus neoformans

An understanding of the connections between metabolism and elaboration of virulence factors during host colonization by the human-pathogenic fungus Cryptococcus neoformans is important for developing antifungal therapies. Lipids are abundant in host tissues, and fungal pathogens in the phylum basidiomycota possess both peroxisomal and mitochondrial β-oxidation pathways to utilize this potential carbon source. In addition, lipids are important signaling molecules in both fungi and mammals. In this report, we demonstrate that defects in the peroxisomal and mitochondrial β-oxidation pathways influence the growth of C. neoformans on fatty acids as well as the virulence of the fungus in a mouse inhalation model of cryptococcosis. Disease attenuation may be due to the cumulative influence of altered carbon source acquisition or processing, interference with secretion, changes in cell wall integrity, and an observed defect in capsule production for the peroxisomal mutant. Altered capsule elaboration in the context of a β-oxidation defect was unexpected but is particularly important because this trait is a major virulence factor for C. neoformans. Additionally, analysis of mutants in the peroxisomal pathway revealed a growth-promoting activity for C. neoformans, and subsequent work identified oleic acid and biotin as candidates for such factors. Overall, this study reveals that β-oxidation influences virulence in C. neoformans by multiple mechanisms that likely include contributions to carbon source acquisition and virulence factor elaboration.     

In vitro susceptibility characteristics of Cryptococcus neoformans varieties from AIDS patients in Goiânia, Brazil

Sixty clinical isolates of Cryptococcus neoformans from AIDS from Goiania, state of Goiás, Brazil, were characterized according to varieties, serotypes and tested for antifungal susceptibility. To differentiate the two varieties was used L-canavanine-glycine-bromothymol blue medium and to separate the serotypes was used slide agglutination test with Crypto Check Iatron. The Minimal Inhibitory Concentration (MIC) of fluconazole, itraconazole, and amphotericin B were determined by the National Committee for Clinical Laboratory Standards macrodilution method. Our results identified 56 isolates as C. neoformans var. neoformans serotype A and 4 isolates as C. neoformans var. gattii serotype B. MIC values for C. neoformans var. gattii were higher than C. neoformans var. neoformans. We verified that none isolate was resistant to itraconazole and to amphotericin B, but one C. neoformans var. neoformans and three C. neoformans var. gattii isolates were resistant to fluconazole. The presence of C. neoformans var. gattii fluconazole resistant indicates the importance of determining not only the variety of C. neoformans infecting the patients but also measuring the MIC of the isolate in order to properly orient treatment.     

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.     

First isolation and molecular characterization of Cryptococcus neoformans var. grubii in excreta of birds in the urban perimeter of the Municipality of Popayán,Colombia

Background

Cryptococcosis is a systemic opportunistic mycosis, caused by Cryptococcus neoformans and Cryptococcus gattii, which affects mainly the central nervous system of immunocompromised patients; no reports have been made on the isolation of the fungus from the environment of Popayán, Colombia.

白地霉Cryytococcus neoformans脂肪酶产酶条件与纯化

目的:获得白地霉脂肪酶发酵的最优条件。方法:采用摇床培养的方法对各种影响因素进行筛选,结果:最适产酶条件为:豆油作碳源,蛋白胨作氮源,PEG600为表面活性剂,添加MgSO4、FeSO4、ZnSO4,pH8.7培养48h。采用双水相萃取和凝胶过滤两步纯化得到电泳均一的脂肪酶,纯化倍数达到72.2倍,其相对分子量约为38±1kDa。结论:得出采用双水相萃取脂肪酶的方法比较理想,该研究为新疆天山一号冰川白地霉脂肪酶的研究奠定了理论基础。     

Carbonic anhydrase inhibitors. Inhibition of the fungal β-carbonic anhydrases from Candida albicans and Cryptococcus neoformans with boronic acids

Inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with a series of aromatic, arylalkenyl- and arylalkylboronic acids was investigated. Aromatic, 4-phenylsubstituted- and 2-naphthylboronic acids were the best Can2 inhibitors, with inhibition constants in the range of 8.5–11.5 μM, whereas arylalkenyl and aryalkylboronic acids showed K_Is in the range of 428–3040 μM. Nce103 showed a similar inhibition profile, with the 4-phenylsubstituted- and 2-naphthylboronic acids possessing K_Is in the range of 7.8–42.3 μM, whereas the arylalkenyl and aryalkylboronic acids were weaker inhibitors (K_Is of 412–5210 μM). The host human enzymes CA I and II were also effectively inhibited by these boronic acids. The B(OH)₂ moiety is thus a new zinc-binding group for designing effective inhibitors of the α- and β-CAs.     

Polyamine depletion and growth inhibition ofCryptococcus neoformans by α-difluoromethylornithine and cyclohexylamine

The ability of two known inhibitors of polyamine synthesis,-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and cyclohexylamine, an inhibitor of spermidine synthase, to inhibit thein vitro growth and polyamine synthesis of clinical isolates ofCryptococcus neoformans was examined. Treatment ofC. neoformans with either DFMO or cyclohexylamine resulted in depletion of cellular polyamines and inhibition of growth.Cryptococcus neoformans was shown to lack detectable spermine and to require high concentrations of spermidine, but not putrescine, for growth. The growth inhibition by DFMO and cyclohexylamine was reversed by exogenous polyamines. These findings document the ability of cyclohexylamine and DFMO to inhibit polyamine synthesis and growth in clinically important isolates ofC. neoformans.     

Isolation and Characterization of Cryptococcus neoformans Varieties Recovered from Natural Sources in Bogotá, Colombia, and Study of Ecological Conditions in the Area

Cryptococcus neoformans, the etiological agent of cryptococcosis, has been associated with avian droppings and certain trees in different countries, including Colombia. C neoformans environmental isolates were obtained in urban areas in Bogotá, Colombia, and the strains recovered were phenotypically characterized. Attempts to determine the ecological conditions (micro- and macroclimatic) possibly related to their habitat were also undertaken. Four hundred and eighty samples from bark, soil around trunk bases, and detritus inside hollows of 32 trees were collected in three urban areas during a 5-month period, as well as 89 avian droppings samples from different places. Of plant samples, 6.7% collected from nine tree species yielded C. neoformans var. gattii, serotype B strains in 99% of the cases, and C. neoformans var. grubii, serotype A in 1%. The yeast was more frequently recovered from bark than from soil or detritus inside hollows, and from trees with hollows or rotted wood rather than from trees in which birds nest. C. neoformans was present with higher frequency and density in the rainy season than in the dry season; we found that slightly higher temperature and humidity values of the microhabitat, as compared to those of the environment, favored fungal occurrence, but the phenological state of the tree did not. Of dropping samples, 7.9% yielded C. neoformans strains, all of them C. neoformans var. grubii, serotype A. The yeast was obtained more frequently from dry droppings than from moist ones, but neither the sunlight exposure nor the site of collection of samples was correlated with this occurrence. Population density was significantly higher in droppings than in tree samples. Under laboratory conditions, isolates of different serotype showed similar capsular sizes. Water content and pH ranges were wide and did not show any significant difference between positive and negative samples.     

条件致病真菌新型隐球酵母（Cryptococcus neoformans）致病性研究进展

摘要：真菌感染引起的疾病在临床上呈现较快的上升趋势,在国际上引起了广泛的重视。新型隐球酵母是主要病原真菌之一。过去十余年,对其致病的分子生物学研究有了较大的进展。重要致病因子（virulence factors）的合成调控及致病过程中信号传导途径等研究进展较快,这些都可能成为治疗甚至预防该真菌的焦点。本文在简要介绍致病因子的基础上,详细综述了分子生物学研究的进展情况。     

Do major species concepts support one, two or more species within Cryptococcus neoformans?

Cryptococcus neoformans, the agent of cryptococcosis, had been considered a homogeneous species until 1949 when the existence of four serotypes was revealed based on the antigenic properties of its polysaccharide capsule. Such heterogeneity of the species, however, remained obscure until the two morphologically distinct teleomorphs of C. neoformans were discovered during the mid 1970s. The teleomorph Filobasidiella neoformans was found to be produced by strains of serotype A and D, and Filobasidiella bacillispora was found to be produced by strains of serotype B and C. Ensuing studies revealed numerous differences between the anamorphs of the two Filobasidiella species with regard to their ecology, epidemiology, pathobiology, biochemistry and genetics. At present, the etiologic agent of cryptococcosis is classified into two species, C. neoformans (serotypes A and D) and Cryptococcus gattii (serotypes B and C). Intraspecific genetic diversity has also been revealed as more genotyping methods have been applied for each serotype. As a result, the number of scientifically valid species within C. neoformans has become a controversial issue because of the differing opinions among taxonomists as to the appropriate definition of a species. There are three major species concepts that govern classification of organisms: phenetic (morphologic, phenotypic), biologic (interbreeding) and cladistic (evolutionary, phylogenetic). Classification of the two C. neoformans species has been based on the phenetic as well as the biologic species concept, which is also supported by the cladistic species concept. In this paper, we review and attest to the validity of the current two-species system in light of the three major species concepts.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with aliphatic and aromatic carboxylates

The inhibition of the β-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with carboxylates such as the C1–C5 aliphatic carboxylates, oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and some benzoates has been investigated. The best Can2 inhibitors were acetate and maleate (K_Is of 7.3–8.7 μM), whereas formate, acetate, valerate, oxalate, maleate, citrate and 2,3,5,6-tetrafluorobenzoate showed less effective inhibition, with K_Is in the range of 42.8–88.6 μM. Propionate, butyrate, malonate, l-malate, pyruvate, l-lactate and benzoate, were weak Can2 inhibitors, with inhibition constants in the range of 225–1267 μM. Nce103 was more susceptible to inhibition with carboxylates compared to Can2, with the best inhibitors (maleate, benzoate, butyrate and malonate) showing K_Is in the range of 8.6–26.9 μM. l-Malate and pyruvate together with valerate were the less efficient Nce103 inhibitors (K_Is of 87.7–94.0 μM), while the remaining carboxylates showed a compact behavior of efficient inhibitors (K_Is in the range of 35.1–61.6 μM). Notably the inhibition profiles of the two fungal β-CAs was very different from that of the ubiquitous host enzyme hCA II (belonging to the α-CA family), with maleate showing selectivity ratios of 113.6 and 115 for Can2 and Nce103, respectively, over hCA II inhibition. Therefore, maleate is a promising starting lead molecule for the development of better, low nanomolar, selective β-CA inhibitors.     

Noncanonical Gβ Gib2 Is a Scaffolding Protein Promoting cAMP Signaling through Functions of Ras1 and Cac1 Proteins in Cryptococcus neoformans

Gβ-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gβ Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.     

Benzenesulfonamides incorporating nitrogenous bases show effective inhibition of β-carbonic anhydrases from the pathogenic fungi Cryptococcus neoformans,Candida glabrata and Malassezia globosa

There is an urgent need for new chemotherapic agents to treat human fungal infections due to emerging and spreading globally resistance mechanisms. Among the new targets that have been recently investigated for the development of antifungal drugs there are the metallo-enzymes Carbonic Anhydrases (CAs, EC 4.2.1.1). The inhibition of the β-CAs identified in many pathogenic fungi leads to an impairment of parasite growth and virulence, which in turn leads to a significant anti-infective effect. Based on antifungal nucleoside antibiotics, the inhibition of the β-CAs from the resistance-showing fungi Candida glabrata (CgNce103), Cryptococcus neoformans (Can2) and Malasszia globosa (MgCA) with a series of benzenesulfonamides bearing nitrogenous bases, such as uracil and adenine, is here reported. Many such compounds display low nanomolar (<100 nM) inhibitory potency against Can2 and CgNce103, whereas the activity of MgCA is considerably less affected (inhibition constants in the range 138.8–5601.5 nM). The β-CAs inhibitory data were compared with those against α-class human ubiquitous isoforms. Interesting selective inhibitory activities for the target fungal CAs over hCA I and II were reported, which make nitrogenous base benzenesulfonamides interesting tools and leads for further investigations in search of new antifungal with innovative mechanisms of action.