Mycelial Phase Research Articles

VEA1 is required for cleistothecial formation and virulence in Histoplasma capsulatum

Histoplasma capsulatum is a pathogenic fungus dependent on dimorphism for virulence. Among the four described Velvet family genes, two of them, Ryp2 and Ryp3, have been shown to be required for dimorphism. It is known that Velvet A (VeA) is necessary for sexual development and toxin production in Aspergillus nidulans. However, the role of the VeA ortholog in H. capsulatum has not yet been explored. Vea1, H. capsulatum homolog of VeA, was studied to determine its role in cleistothecial formation, dimorphism, and virulence. H. capsulatum Vea1 restores cleistothecial formation and partially restores sterigmatocystin production in an A. nidulans veA deletion strain. Furthermore, silencing VEA1 in an H. capsulatum strain capable of forming cleistothecia abolishes cleistothecial formation. Silenced strains also switch to mycelial phase faster, and show impaired switching to the yeast phase once in mycelial phase. Virulence in mice and macrophages is attenuated in VEA1 silenced strains and silenced strains demonstrate increased sensitivity during growth under acidic conditions. These results indicate that H. capsulatum Vea1 shares a similar role in development as VeA. H. capsulatum is also more susceptible to growth in acidic conditions when VEA1 is silenced, which may contribute to the silenced strains’ attenuated virulence in mice and macrophages.

Secretome characteristics of pelletized Trichoderma reesei and cellulase production

Trichoderma reesei is an important cellulase producer and its secondary mycelial phase is responsible for cellulase expression and secretion in submerged fermentation. Little is known regarding the effects of fungal morphology on cellulase production by Trichoderma sp. In this study we aimed to extend the understanding of cellulase production by T. reesei, especially correlating cellulase productivity with pellet morphology and with its secretome characteristics. We found that T. reesei was more likely to form pellets in malt extract broth than in potato dextrose broth. CaCO(3) helped in formation of fine pellets in malt extract broth. 10(9) spores/ml resulted in formation of pellets with the size of 0.13 ± 0.047 mm. LC/MS spectrometry analysis indicated that the secretomes from pellet was different from that of mycelial mat under the same fermentation conditions. Optimization tests showed that lactose, xylose and Pluronic F68 are important for efficient production of cellulases with FPU activity in the pellets fermentation. This is the first report on the artificial formation of pellets by Trichoderma sp. as well as correlation between physiological characteristic of the pellets and cellulase production by T. reesei. The findings from this study can be used for improvement of cellulase productivity.

Distinct chitinases are expressed during various growth phases of the human pathogen Paracoccidioides brasiliensis

The aim of this work was the partial purification and subsequent evaluation of chitinase expression during the various growth phases of Paracoccidioides brasiliensis. Initially, PbCTS1r was expressed as a recombinant protein and displayed enzymatic activity against 4-MU-[N-acetylglucosamine (GlcNAc)]3 and 4-MU-(GlcNAc)2. Two proteins, 45 kDa and 39 kDa in size, were partially purified from P. brasiliensis yeast crude extract using cation-exchange chromatography coupled with HPLC and were characterised as PbCTS1 and PbCTS2, respectively. Anti-PbCTS1r antibody recognised two proteins in the crude extracts of yeast and the transitional stage between mycelial and yeast phases. In crude extracts of mycelium, only the 45 kDa protein was detected. However, quantitative real-time polymerase chain reaction led to the detection of small quantities of Pbcts2 transcript in the mycelial phase. In the yeast cell wall extract, only the 39 kDa protein was detected. Moreover, both proteins were secreted by the yeast parasitic phase, suggesting that these proteins participate in the modulation of the fungal environment. Phylogenetic analysis of the predicted PbCTS1 and PbCTS2 proteins indicated that they code for distinct chitinases in P. brasiliensis. During evolution, P. brasiliensis could have acquired the paralogues Pbcts1 and Pbcts2 for growth and survival in diverse environments in both saprophytic and parasitic phases.

Cell‐free antigens of Sporothrix brasiliensis: antigenic diversity and application in an immunoblot assay

Sporotrichosis is a subcutaneous mycosis diagnosed by isolation of the fungus in culture. Serological tests for help in diagnosis in general do not use purified or recombinant antigens, because there is a paucity of described immunoreactive proteins, especially for the new described Sporothrix species, such as Sporothrix brasiliensis. This study aims to characterise antigens from S. brasiliensis and verify their application in serodiagnosis of sporotrichosis. An immunoblot assay allied with computer-based analysis was used to identify putative antigenic molecules in a cell-free extracts of both morphological phases of this fungus, and to delineate antigenic polymorphism among seven S. brasiliensis isolates and one S. schenckii Brazilian strain. The mycelial and yeast phase of the fungus originated 14 and 23 reactive bands, respectively, which were variable in intensity. An 85 kDa antigen, verified in the yeast phase of the fungus, was observed in all strains used and the immunodominant protein was identified. This protein, however, cross-react with serum samples from patients infected with other pathogens. The results show that the S. brasiliensis cell-free antigen extract is a single and inexpensive source of antigens, and can be applied on the sporotrichosis serodiagnosis.

Conserved regulators of the cell separation process in Schizosaccharomyces

The fission yeasts (Schizosaccharomyces) representing a highly divergent phylogenetic branch of Fungi evolved from filamentous ancestors by gradual transition from mycelial growth to yeast morphology. For the transition, a mechanism had been developed that separates the sister cells after the completion of cytokinesis. Numerous components of the separation mechanism have been characterised in Schizosaccharomycespombe, including the zinc-finger transcription factor Ace2p and the fork-head transcription factor Sep1p. Here we show that both regulators have regions conserved within the genus. The most conserved parts contain the DNA-binding domains whose amino-acid sequences perfectly reflect the phylogenetic positions of the species. The less conserved parts of the proteins contain sequence blocks specific for the whole genus or only for the species propagating predominantly or exclusively as yeasts. Inactivation of either gene in the dimorphic species Schizosaccharomycesjaponicus abolished cell separation in the yeast phase conferring hypha-like morphology but did not change the growth pattern to unipolar and did not cause extensive polar vacuolation characteristic of the true mycelium. Neither mutation affected the mycelial phase, but both mutations hampered the hyphal fragmentation at the mycelium-to-yeast transition. Ace2pSj acts downstream of Sep1pSj and regulates the orthologues of the Ace2p-dependent S.pombe genes agn1+ (1,3-alpha-glucanase) and eng1+ (1,3-beta-glucanase) but does not regulate the orthologue of cfh4+ (chitin synthase regulatory factor). These results and the complementation of the cell separation defects of the ace2− and sep1− mutations of S.pombe by heterologously expressed ace2Sj and sep1Sj indicate that the cell separation mechanism is conserved in the Schizosaccharomyces genus.

DESENVOLVIMENTO MICELIAL IN VITRO DE PLEUROTUS SP. EM PALHA DE ARROZ SUPLEMENTADA COM SERRAGEM DE COURO

RESUMO O presente trabalho foi desenvolvido com o objetivo de avaliar a produção de biomassa, crescimento micelial e a colonização do substrato palha de arroz por Pleurotus ostreatus e Pleurotus ostreatoroseus, sob o efeito da adição de serragem de couro curtida ao tanino vegetal. O experimento 1 constituiu-se na formulação de meios de cultura, à base de palha de arroz adicionada de serragem de couro, nas concentrações 0, 5, 10, 15, 20, 25 e 30%. Discos de micélio de P. ostreatus (BF24) e P. ostreatoroseus (POR01/06) foram transferidos para o centro de placas contendo meios previamente preparados. Eles foram incubados a 28º C. No segundo experimento, o substrato palha de arroz foi submetido as mesmas condições experimentais acima descritas. O substrato inoculado com as duas cepas foi acondicionado em tubos de ensaio e incubado a 28º C. A biomassa fúngica foi avaliada pela diferença de massa seca e massa úmida, enquanto que para o crescimento do micélio realizaram-se mensurações em períodos pré-determinados até a completa colonização do meio ou do substrato. Verificou-se que os tratamentos com adição de 5, 15 e 25% e 15% deste suplemento para P. ostreatus proporcionaram redução da biomassa e do crescimento micelial, respectivamente. Na fase de miceliação o acréscimo de 5% deste suplemento a palha de arroz apresentou efeito positivo para o desenvolvimento de P. ostreatus.

Evaluación de la técnica PCR anidada para el diagnóstico de la esporotricosis experimental

BackgroundSporotrichosis caused by the dimorphic fungus Sporothrix schenckii can presents in a variety of clinical forms. Routine diagnosis is made by mycology and serology studies. Few investigations have been focused on the evaluation of the molecular diagnosis. AimTo determine the value of the nested PCR technique for the diagnosis of experimental sporotrichosis in organs of mice, and to compare the results with the established laboratory diagnostic procedures. MethodsBALB/c mice were inoculated with growing concentrations of the 2 morphological phases of the fungus. The infected animals were sacrificed one month later and specimens from liver, spleen, lung and testicle were obtained to perform wet mount, culture and molecular diagnosis by the nested PCR technique. Blood samples were obtained for determination of specific antibodies against S. schenckii by the double immunodiffusion procedure. ResultsThe pathogenicity observed with the different concentrations of the fungus inoculated and its isolation by culture, showed scarce differences in the study of specimens from organs infected with the 2 morphological phases of S. schenckii. Specimens from organs of mice inoculated with the mycelial phase when studied by wet mount and culture, showed a higher positivity (100 and 37.5%) than those from mice inoculated with the yeast phase (73 and 2%). However, diagnosis by the nested PCR molecular technique applied to the latter specimens showed a higher percentage of positivity (75%) and 43% of positive results coming from animals infected with the mycelial phase. Specific antibody detection was positive in 100% all groups of infected mice. ConclusionsIn the study of experimental sporotrichosis in mice, the culture, as well as the antibody detection, was an effective diagnostic procedure, while the nested PCR and microscopic studies had a lower diagnostic value.

LIMBAH GERGAJI KAYU SEBAGAI BAHAN FORMULA MEDIA JAMUR SHIITAKE (LENTINULA EDODES)

The sawdust can be utilized as mushroom formulation medium , like shiitake (Lentinus edodes /Lentinula edodes). The antitumor properties of lentinus are attributed to the presence of polysaccharide lentinan. Lentinan can be utilized as reducing diabetes, and effective to againts influenza virus. Usually, Albazia falcataria sawdust were utilized by mushroom growers in Indonesia. Optimum temperature for shiitake growth is 24oC and pH4.7 – 4.8 (mycelium phase), 20oC and pH 4.2 – 4.6.(fruit body phase). The elevation for best cultivation is 800 – 1000 m above sea level, humudity almost 95 – 100%. Ifused log system, shiitake will be harvested after 2-3 months fruiting bodies were produced. Biological efficiency (BER) was 45%.

Ultrastructural study of the mycelial phase of clinical isolates of Sporothrix schenckii obtained from feline, canine and human cases of sporotrichosis

Using transmission electron microscopy, we studied the presence of melanin and cell wall thickness of clinical isolates of Sporothrix schenckii obtained from cats, dogs and humans as compared to reference strains. We detected differences regarding presence of the melanin among the clinical isolates of S. schenckii and a correlation between presence of melanin and cell wall thickness.

Protein Profiling of the Dimorphic Pathogenic Fungus, Sporothrix schenckii

Sporotrichosis is a common cutaneous mycosis caused by the dimorphic fungus Sporothrix schenckii, which exhibits a temperature-dependent dimorphic switch. At 25°C, it grows in a mycelial phase, while at 37°C, it forms unicellular yeast cells. The formation of yeast cells was thought to be a requisite for the pathogenicity of S. schenckii. To identify fragments that might be related to morphogenesis, whole-cell proteins from the mold and early yeast stages of S. schenckii were analyzed using 2DE. Among thousands of protein molecules displayed, more than 300 showed a differential expression between the two phases. In particular, 24 yeast-specific proteins were identified using MALDI-TOF/MS. One of the most interesting proteins was a hybrid histidine kinase, DRK1, a global regulator of dimorphism and virulence in Blastomyces dermatitidis and Histoplasma capsulatum that was abundant in the yeast phase. Our study introduced a new approach to study dimorphism in S. schenckii, and the data may help us better understand the molecular mechanisms of phase transition.

Ultrastructural study of the mycelial phase of clinical isolates of Sporothrix schenckii obtained from feline, canine and human cases of sporotrichosis.

Using transmission electron microscopy, we studied the presence of melanin and cell wall thickness of clinical isolates of Sporothrix schenckii obtained from cats, dogs and humans as compared to reference strains. We detected differences regarding presence of the melanin among the clinical isolates of S. schenckii and a correlation between presence of melanin and cell wall thickness.

Identificación rápida de Histoplasma capsulatum en lisados de cultivo

Background Histoplasma capsulatum is the agent of histoplasmosis, a deep mycosis mainly afflicting immunocompromised patients. Rapid identification of the fungus isolated from clinical specimens allows timely administration of specific treatment. Aim To assess the ability of a dual PCR system targeting specific H. capsulatum DNA sites to identify fungal species in simple aqueous lysates from cultured fungi. Methods We analysed the performance of two independent PCR reactions designed to amplify fragments of 111 and 279 bp included in H. capsulatum-specific gene AgM. We used 248 H. capsulatum strains and 68 isolates of other fungal species. Reaction templates consisted of aqueous lysates of cultured fungi (either in mycelial or yeast phase) obtained after three cycles of boiling and immediate cooling at 0 °C. Selected strains were submitted to conventional DNA extraction and/or sequencing. Results Both PCR systems performed identically. Amplification from aqueous lysates was achieved from 239 H. capsulatum strains; the remaining 9 strains only showed specific bands when purified DNA was used as template. Of all other fungal species tested, only 2 Emmonsia crescens strains amplified H. capsulatum-specific bands and sequences of their amplified PCR products matched > 97% with H. capsulatum sequences. Total test time did not exceed 7 h with 96% sensitivity, 97% specificity and 99% positive predictive value. Conclusions The assay is fast, accurate and economical, and can be an alternative method for presumptive identification of H. capsulatum in simple culture lysates.

Distribución de las especies de Malassezia en pacientes con pitiriasis versicolor y en individuos sanos

Purpose This study analyses the prevalence of Malassezia species in the skin lesions of patients with pityriasis versicolor and in the healthy skin of a healthy individual group. Materials and Methods The group studied included one hundred and fifty patients with pityriasis versicolor. Samples were taken from the pityriasis versicolor lesions by means of Sellotape for direct microscopy with KOH + Parker ink, and scales were obtained by scarping for culture, and inoculated on modified Dixon agar plates. Samples were also taken of healthy skin of the forehead and of the shoulders of one hundred and fifty healthy individual group. All these samples were used for culture and inoculated on the medium, but were not examined by direct microscopy. The yeasts were identified by their morphological and physiological characteristics. Results M. globosa was isolated in 130 cases (86,7%) of 150 patients with pityriasis versicolor. M. sympodialis was also found in 63 cases (42%). In healthy skin of the forehead of healthy individuals a total of 23 isolates from the 150 samples were obtained. M. sympodialis was the predominant species (56,52%), followed by M. globosa (47,8%). In healthy skin of the shoulders a total of 91 isolates were obtained. M. sympodialis was by far the predominant species (91,2%) followed by M. globosa (7,6%) and M. slooffiae (1,09%). Conclusions M. globosa, in its mycelial phase, is the predominant species in the ethiology of pityriasis versicolor.

Brote de histoplasmosis en la Escuela de Cadetes de la Base Aérea de Morón, Provincia de Buenos Aires, República Argentina

A histoplasmosis outbreak affecting 6 previously healthy Air Force cadets is herein presented. The patients suffered from fever and respiratory symptoms after having cleaned an abandoned hangar soiled with pigeons and bat droppings. They all presented fever, myalgia, tachypnea, and nonproductive cough. Chest X-ray and CT scan studies showed disseminated reticulonodular images affecting both lungs. Hiliar adenomegalies were also observed. All patients achieved a favourable outcome without antifungal treatment. Both serologic tests searching for specificic antibodies (immunodiffusion and counterimmunoelectrophoresis) and histoplasmin skin tests were positive in all cases. Five soil samples mixed with pigeons and bat droppings were collected from the hangar. Suspensions of these samples were inoculated into 20 hamsters by intraperitoneal injection; mycelial phase of H. capsulatum was isolated from liver and spleen cultures. The genetic profile of this strain was compared with 12 isolates obtained from Argentinean patients, and a great degree of homogeneity was observed (> 96% similarity). Although histoplasmosis is endemic in the wet Pampas, this is the first epidemic outbreak reported south of the 34th parallel.

1,3-β- d-Glucan synthase of Paracoccidioides brasiliensis: recombinant protein, expression and cytolocalization in the yeast and mycelium phases

Paracoccidioides brasiliensis is a thermo-dimorphic human pathogenic fungus that in the mycelium phase lives at 23 °C in environment and in the yeast phase at 37 °C in the host tissues. In P. brasiliensis, the main polymers that compound the cell wall are chitin, 1,3-β- d-glucan and 1,3-α-glucan. They make a primary barrier responsible for the structural integrity and form of the cell wall. In P. brasiliensis, just one homologue of 1,3-β- d-glucan synthase gene ( PbFKS1) was found. Here, the active recombinant protein ( PbFks1pc) containing the catalytic region was obtained in Escherichia coli. In addition, a paradoxical dissociation was detected between the expression of the PbFKS1 transcript and the level of the corresponding protein PbFks1p, which was higher in the yeast phase, versus the amount of 1,3-β- d-glucan polymer, which was higher in the mycelium phase. Western blot analysis using protein extracts of cellular fractions showed that PbFks1p is present in the membrane-enriched fraction of mycelium and yeast cells and in the cell wall-enriched fractions of yeast cells. Confocal-immunocytolocalization of PbFks1p identified the protein in the apical growing region of the mycelium and distributed on the surface of the yeast cell. Two possible mechanisms could explain the above-mentioned discrepancy between the data: (a) overexpression of Rho1 GTPase as a regulator of 1,3-β- d-glucan synthase; (b) possible post-translational regulation of PbFks1p in P. brasiliensis isolates.

Enhanced hexadecane degradation and low biomass production by Aspergillus niger exposed to an electric current in a model system

The effects of an electric current on growth and hexadecane (HXD) degradation by Aspergillus niger growth were determined. A 450-mL electrochemical cell with titanium ruthenium-oxide coated electrodes and packed with 15 g of perlite (inert biomass support) was inoculated with A. niger (2.0×10(7) spores (g of dry inert support)(-1)) and incubated for 12 days (30 °C; constant ventilation). 4.5 days after starting culture a current of 0.42 mA cm(-2) was applied for 24h. The current reduced (52±11%) growth of the culture as compared to that of a culture not exposed to current. However, HXD degradation was 96±1.4% after 8 days whereas it was 81±1.2% after 12 days in control cultures. Carbon balances of cultures not exposed to current suggested an assimilative metabolism, but a non-assimilative metabolism when the current was applied. This change can be related to an increase in total ATP content. The study contributes to the knowledge on the effects of current on the mycelial growth phase of A. niger, and suggests the possibility of manipulating the metabolism of this organism with electric current.

Differential expression ofCandida dubliniensis-secreted aspartyl proteinase genes (CdSAP1–4) under different physiological conditions and during infection of a keratinocyte culture

The in vitro and keratinocyte (HaCAT cells) culture expression of four putative genes coding for secreted aspartyl proteases of Candida dubliniensis-CdSAP1, CdSAP2, CdSAP3, and CdSAP4 (CdSAP1-4) - is reported for the first time. In addition, CdSAP7, 8, 9, and 10, orthologous genes of Candida albicans, were recognized in C. dubliniensis genome. There are no orthologs of C. albicans SAP5 and 6 in C. dubliniensis. The expression of CdSAP1 and 2 was independent of the morphological stage of C. dubliniensis; they are expressed at both pH 4 and pH 7, and were induced with albumin as nitrogen source. CdSAP3 expression was regulated by the pH, and was related to the infection process of keratinocytes. Expression of CdSAP4 predominated during the mycelial phase and the initial stage of keratinocyte infection. During infection of the HaCaT cell line, only genes CdSAP3-4 were expressed, and keratinocytes were affected in their number and shape by the infection with C. dubliniensis; however, this effect decreased in the presence of pepstatin A (aspartyl protease inhibitor). Pepstatin A was not able to inhibit keratinocyte damage. Based on the aforementioned, we suggest that the Saps from C. dubliniensis could be considered a virulence factor just as those from C. albicans, and participants in the nitrogen metabolism of the yeast for nutrient acquisition.

Transcription levels of CHS5 and CHS4 genes in Paracoccidioides brasiliensis mycelial phase, respond to alterations in external osmolarity, oxidative stress and glucose concentration

The complete sequence of Paracoccidioides brasiliensis CHS5 gene, encoding a putative chitin synthase revealed a 5583 nt open reading frame, interrupted by three introns of 82, 87 and 97 bp (GenBank Accession No EF654132). The deduced protein contains 1861 amino acids with a predicted molecular weight of 206.9 kDa. Both its large size and the presence of a N-terminal region of approx. 800 residues with a characteristic putative myosin motor-like domain, allow us to include PbrChs5 into class V fungal chitin synthases. Sequence analysis of over 4 kb from the 5′ UTR region in CHS5, revealed the presence of a previously reported CHS4 gene in P. brasiliensis, arranged in a head-to-head configuration with CHS5. A motif search in this shared region showed the presence of stress response elements (STREs), three binding sites for the transcription activators Rlm1p (known to be stimulated by hypo-osmotic stress) and clusters of Adr1 (related to glucose repression). A quantitative RT-PCR analysis pointed to changes in transcription levels for both genes following oxidative stress, alteration of external osmolarity and under glucose-repressible conditions, suggesting a common regulatory mechanism of transcription.

Adiaspiromycosis Due to Emmonsia crescens is Widespread in Native British Mammals

Adiaspiromycosis caused by Emmonsia crescens is primarily a respiratory disease affecting small mammals, especially members of the Families Rodentia, Carnivora and Mustelidae. Although isolated reports exist of adiaspiromycosis in free-living British wildlife, the extent of infection in wild animals in the UK, and the significance of any associated pathology are unclear. Here, we report the results of histopathological examination of lungs of free-living wild mammals from the south-west UK coupled with digestion of lung material in potassium hydroxide followed by centrifugation and microscopic examination for the presence of adiaspores. The combined results showed that almost one-third (27/94, 28.7%) of animals examined had evidence of infection with E. crescens. Attempts to culture E. crescens from infected lungs were unsuccessful. However, E. crescens could be confirmed as the causative agent by PCR amplification and sequencing of DNA from adiaspores micro-dissected from animal lungs. The prevalence of adiaspiromycosis was largely independent of animal species or precise geography. Adiaspore burdens in most animals were low, consistent with transient exposure to E. crescens. However, burdens in several animals suggested heavy or repeated exposures to E. crescens, and were considered sufficient to have significantly impaired respiratory function. Finally, since E. crescens is apparently widespread in UK mammals and the first UK human case of adiaspiromycosis was reported recently, we present data obtained using a previous isolate of E. crescens demonstrating that both the mycelial and adiaspore phases of the organism are susceptible to amphotericin B, voriconazole, itraconazole and caspofungin.

The interaction between Histoplasma capsulatum cell wall carbohydrates and host components: relevance in the immunomodulatory role of histoplasmosis

Histoplasma capsulatum is an intracellular fungal pathogen that causes respiratory and systemic disease by proliferating within phagocytic cells. The binding of H. capsulatum to phagocytes may be mediated by the pathogen's cell wall carbohydrates, glucans, which consist of glucose homo and hetero-polymers and whose glycosydic linkage types differ between the yeast and mycelial phases. The alpha-1,3-glucan is considered relevant for H. capsulatum virulence, whereas the beta-1,3-glucan is antigenic and participates in the modulation of the host immune response. H. capsulatum cell wall components with lectin-like activity seem to interact with the host cell surface, while host membrane lectin-like receptors can recognize a particular fungal carbohydrate ligand. This review emphasizes the relevance of the main H. capsulatum and host carbohydrate-driven interactions that allow for binding and internalization of the fungal cell into phagocytes and its subsequent avoidance of intracellular elimination.