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PubMed2006

Extracellular peptidase in the fungal pathogen Pseudallescheria boydii.

Pseudallescheria boydii is a ubiquitous filamentous fungus capable of causing invasive disease in humans. In the present study, using sodium dodecyl sulfate-polyacrylamide gels containing bovine serum albumin as co-polymerized substrate, we identified a 28-kDa proteolytic activity released to the extracellular environment by mycelia of P. boydii. This peptidase was detected during the growth of P. boydii in Sabouraud-dextrose medium for 13 days and reached its maximal production on day 7. The 28-kDa peptidase was active in acidic pH (5.5) and had its activity completely blocked by 1,10-phenanthroline, a potent zinc-metallopeptidase inhibitor. Two other metallopeptidase inhibitors, EDTA and EGTA, were also tested and no alterations were observed in the activity of the 28-kDa extracellular peptidase. Likewise, E-64 (a cysteine peptidase inhibitor), phenylmethylsulphonyl fluoride (a serine peptidase inhibitor), and pepstatin A (an aspartyl peptidase inhibitor) did not significantly alter the enzymatic behavior. Collectively, we described for the first time the expression of an extracellular metallopeptidase in the human opportunistic fungal pathogen P. boydii.

Enzyme Activation↗
PubMed2002

A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene.

Aspergillus fumigatus is an important opportunistic human pathogenic fungus. In severely immunocompromised patients, the fungus causes life-threatening diseases, such as pneumonia and invasive aspergillosis. In order to obtain a better understanding of the key elements involved in A. fumigatus virulence and for identifying possible drug targets, it is essential to be able to generate gene-deletion strains. Until recently, the molecular techniques available did not provide a rapid method for gene deletion. A novel method described for A. nidulans was adapted for A. fumigatus. This method is quick and produces an increased homologous recombination efficiency. By using an Escherichia coli strain expressing the lambda red operon, it is possible to induce an in vivo recombination of a PCR fragment flanked by >50-bp regions with a cosmid containing the gene of interest. This produces cosmids in which the gene of interest has been replaced by a bi-functional marker. Such cosmids have large flanking regions surrounding the selectable marker pyrG of A. fumigatus used here, which result in high recombination efficiencies in A. fumigatus. Here, we identified a new ABC transporter-encoding gene in A. fumigatus, designated abcA. By using this method, an A. fumigatus knock-out mutant was generated, providing evidence that this method of generating gene deletions can also be used in A. fumigatus and significantly broadens our repertoire of molecular techniques to study A. fumigatus.

ATP-Binding Cassette Transporters↗
PubMed2003

Chitosan improves development, and protects Vitis vinifera L. against Botrytis cinerea.

We evaluated the potential of chitosan both to stimulate plant development and to induce protection from Botrytis cinerea in Vitis vinifera L. plantlets. The presence of 1.75% (v/v) chitogel in the culture medium was the optimal concentration for in vitro grapevine plantlet growth, as determined by measurements on enhancement of root and shoot biomass. Photosynthesis and related parameters were also stimulated in chitogel-treated plantlets. Chitogel reduced the development of Botrytis cinerea and induced cytological alterations to the pathogen. When challenged with the fungus, a significant decrease in disease incidence was observed in plants growing on medium supplemented with chitogel. Furthermore, exogenous foliar applications of chitogel to plantlets growing on chitogel-free medium sensitized them so as to be protected against Botrytis cinerea attack. Our results indicate that chitogel can be used in the vineyard as a means to attain protection against Botrytis cinerea and that its application may counteract the wide use of chemical pesticides.

Botrytis↗
PubMed2026

Genome characterization of two novel mitoviruses and a negative-sense single-stranded RNA mycovirus from the phytopathogenic fungus Clarireedia jacksonii.

Clarireedia jacksonii is a phytopathogenic fungus responsible for dollar spot disease in turfgrass worldwide. In this study, we characterized the complete genome sequences of three novel mycoviruses isolated from C. jacksonii isolate MBCT-836 using next-generation sequencing and the fragmented and primer-ligated dsRNA sequencing (FLDS) method. Two of these viruses, designated Clarireedia jacksonii mitovirus 1 (CjMV1) and Clarireedia jacksonii mitovirus 2 (CjMV2), possess positive-sense single-stranded RNA genomes of 2,575 bp and 2,856 bp, respectively. Both viruses contain a single open reading frame that utilizes the mitochondrial genetic code and encodes an RNA-dependent RNA polymerase (RdRp). Phylogenetic analysis placed CjMV1 and CjMV2 within the genera Unuamitovirus and Duamitovirus, respectively, in the family Mitoviridae. The third virus, Clarireedia jacksonii negative-stranded RNA virus 1 (CjNSV1), features a bisegmented negative-sense RNA genome consisting of a large segment (7,961 nt) encoding an RdRp with a conserved Bunya_RdRp domain, and a small segment (1,444 nt) encoding a protein showing homology to bunyavirus nucleocapsid proteins. Phylogenetic analysis revealed that CjNSV1 clusters with members of the proposed family Sclerobunyaviridae within the order Bunyavirales. To our knowledge, this study provides the first report of complete genome sequences of mycoviruses infecting C. jacksonii, expanding our understanding of the mycovirosphere in economically significant turfgrass pathogens.

Genome, Viral↗
PubMed2004

Central nervous system paracoccidioidomycosis: clinical features and laboratorial findings.

OBJECTIVE: To study prospectively the clinical features and laboratorial characteristics of 24 patients with central nervous system (CNS) involvement with paracoccidioidomycosis (PCM). PCM is an infectious disease caused by the dimorphic fungus Paracoccidioides brasiliensis, endemic in subtropical areas of Central and South America. METHODS: From 173 cases of PCM, 24 (13.9%) had CNS involvement (NPCM) and were studied prospectively from 1993 to 1997. In all the patients, the diagnosis of systemic PCM was made by the demonstration of the P. brasiliensis organisms or positive serology, DID (double immunodiffusion). In seven cases the diagnosis was made by means of a CNS biopsy. CNS clinical manifestations, neuroimaging (CT or MRI) and CSF cytochemical characteristics were reported. RESULTS: The mean age was 44 years (range 25-72 years); 23 patients were male, only one was female. Neurological symptoms began before systemic symptoms in 21%; simultaneously in 33%, and after systemic symptoms in 46%. Epilepsy was the more frequent neurological presentation (44%). Twenty-three cases had parenchymatous involvement and in two of these cases there was an association with meningitis and one case had spinal cord involvement. Lesions were more frequent in the brain hemispheres (69%), in 65% there were multiple granuloma characterized by hypodense images with annular or nodular enhancing. All cases were treated with sulphamethoxazole-trimethoprin. Four patients died, while 20 patients showed a good therapeutic response. CONCLUSION: NPCM should always be considered in the differential diagnosis of expanding lesions of the CNS and meningoencephalitis. Being alert to this diagnosis depends on knowledge of epidemiology. There was good response to sulphamethoxazole-trimethoprin treatment.

Adult↗
PubMed1983

Diagnosis and management of rhino-orbitocerebral mucormycosis (phycomycosis). A report of 16 personally observed cases.

Mucormycosis is the most acutely fatal fungus infection of man. The disease causes a characteristic pattern of clinical symptoms and signs, prompt recognition of which will permit immediate institution of antifungal therapy. Personal experience with 16 cases of the rhino-orbitocerebral form of mucormycosis is the basis of this report. The first of these patients was seen in 1959, and the last in 1981. All of the patients had one or more preexisting diseases, as follows: (1) diabetes mellitus, 13; (2) acute leukemia, 3; (3) terminal carcinomatosis, 1; and (4) chronic sinusitis, 1. The most common initial symptoms and signs were sinusitis, pharyngitis, nasal discharge, and orbital/periorbital pain. Proptosis and formation of a black eschar were only seldom among the initially apparent features. Hyphas were demonstrated in tissue sections in 14 of the 16 patients in whom biopsy was done. Rhizopus species were cultured in 11 of the 13 patients from whom material for culture had been obtained clinically. Five of the 16 patients survived. All of them had been treated with surgical debridement and with intravenous amphotericin B.

Adolescent↗
PubMed2003

Macrophage colony-stimulating factor enhances phagocytosis and oxidative burst of mononuclear phagocytes against Penicillium marneffei conidia.

The responses of rabbit pulmonary alveolar macrophages (PAMs) and elutriated human monocytes (EHMs) to Penicillium marneffei, an emerging dimorphic fungus that may cause fatal disease in human immunodeficiency virus-infected patients, were studied. PAMs and EHMs comparably phagocytosed conidia of two P. marneffei strains in the presence of serum. Electron microscopy showed intraphagosomal destruction of conidia after 12 h. Serum-opsonized conidia elicited significantly more superoxide anion (O(2)(-)) release from EHMs compared to non-opsonized conidia, but equivalent O(2)(-) amounts to that elicited by serum-opsonized Aspergillus fumigatus conidia. Macrophage colony-stimulating factor (M-CSF) significantly enhanced phagocytosis of P. marneffei conidia by PAMs and EHMs, as shown by light microscopy. Moreover, M-CSF enhanced O(2)(-) production by EHMs in response to both serum-opsonized (P<0.001) and non-opsonized (P=0.03) conidia of A. fumigatus as well as conidia of the P. marneffei isolates (P<0.001 and 0.03). We conclude that M-CSF enhances phagocytosis and oxidative metabolism of mononuclear phagocytes suggesting a potential role for this cytokine in host defense against pulmonary and disseminated P. marneffei infection.

Animals↗
PubMed2004

A morphological and cytochemical study of the interaction between Paracoccidiodes brasiliensis and neutrophils.

Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.

Acid Phosphatase↗
PubMed1998

Evidence of idiotypic modulation in the immune response to gp43, the major antigenic component of Paracoccidioides brasiliensis in both mice and humans.

Paracoccidioidomycosis (PCM) is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiologic agent of disease is a thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43,000 D (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with PCM. Gp43 binds to laminin, thus participating in adhesion, invasion and pathogenesis of the fungus. As the role of antibodies in PCM is not fully understood, we decided to investigate the outcome of mice immunization with three distinct anti-gp43 MoAbs (17c, 8a and 24a) coupled with keyhole limpet haemocyanin (KLH). Results show not only the expected presence of anti-Id (AB2) antibodies in the sera of these animals but also a spontaneous and increasing amount of anti-anti-Id (AB3) antibodies after the third course of immunization. Hybridomas producing both AB2 and AB3 MoAbs were obtained using spleen cells from mice immunized with MoAb 17c. AB3 MoAbs were also obtained with spleen cells of mice immunized with MoAbs 8a and 24a. It was also shown that human PCM patients' sera with high titres of anti-gp43 antibodies generate anti-Id antibodies. These data suggest that the immune response to P. brasiliensis can be spontaneously modulated by the idiotypic network.

Animals↗
PubMed2003

Prevalence of Aspergillus fumigatus and other fungal species in the sputum of adult patients with cystic fibrosis.

Aspergillus fumigatus is often found in the respiratory tract secretions of patients with cystic fibrosis (CF), although the role of the fungus for progression of pulmonary disease remains unclear. This study aimed to investigate the frequency of A. fumigatus and other fungi in sputum of adult CF patients using different methods for culture and microscopy. Results from the analysis of 369 samples from 94 patients showed that A. fumigatus could be isolated in 45.7% of patients. Other moulds were rare, but the yeast Candida albicans was another frequent isolate, detected in 75.5% of patients. A comparison of different culture media showed no difference between a selective medium developed to specifically inhibit Pseudomonas aeruginosa and a standard fungal culture medium for growth of A. fumigatus, although both were more efficient for detection of fungi than other bacterial culture media. Fluorescent microscopy with calcofluor white was more sensitive for detection of fungal hyphae in undiluted sputum than standard methylene blue staining. This study shows that A. fumigatus and C. albicans have a high frequency in adult CF patients. Microbiological analysis should routinely include methods for specific identification of fungi to monitor for potential complications arising from fungal disease in these patients.

Adolescent↗
PubMed1989

Host species-specific conservation of a family of repeated DNA sequences in the genome of a fungal plant pathogen.

We have identified a family of dispersed repetitive DNA sequences in the genome of Magnaporthe grisea, the fungus that causes rice blast disease. We have named this family of DNA sequences "MGR" for M. grisea repeat. Analysis of five MGR clones demonstrates that MGR sequences are highly polymorphic. The segregation of MGR sequences in genetic crosses and hybridization of MGR probes to separated, chromosome-size DNA molecules of M. grisea shows that this family of sequences is distributed among the M. grisea chromosomes. MGR sequences also hybridize to discrete poly(A)+ RNAs. Southern blot analysis using a MGR probe can distinguish rice pathogens from various sources. However, MGR sequences are not highly conserved in the genomes of M. grisea field isolates that do not infect rice. These results suggest that host selection for a specific pathogen genotype has occurred during the breeding and cultivation of rice.

Ascomycota↗
PubMed2004

The sphingolipid pathway regulates Pkc1 through the formation of diacylglycerol in Cryptococcus neoformans.

The sphingolipid biosynthetic pathway generates bioactive molecules crucial to the regulation of mammalian and fungal physiological and pathobiological processes. In previous studies (Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R., Hannun, Y. A., and Del Poeta, M. (2001) Genes Dev. 15, 201-212), we demonstrated that an enzyme of the fungal sphingolipid pathway, Ipc1 (inositol-phosphorylceramide synthase-1), regulates melanin, a pigment required for the pathogenic fungus Cryptococcus neoformans to cause disease. In this study, we investigated the mechanism by which Ipc1 regulates melanin production. Because Ipc1 also catalyzes the production of diacylglycerol (DAG), a physiological activator of the classical and novel isoforms of mammalian protein kinase C (PKC), and because it has been suggested that PKC is required for melanogenesis in mammalian cells, we investigated whether Ipc1 regulates melanin in C. neoformans through the production of DAG and the subsequent activation of Pkc1, the fungal homolog of mammalian PKC. The results show that modulation of Ipc1 regulates the levels of DAG in C. neoformans cells. Next, we demonstrated that C. neoformans Pkc1 is a DAG-activated serine/threonine kinase and that the C1 domain of Pkc1 is necessary for this activation. Finally, through both pharmacological and genetic approaches, we found that inhibition of Pkc1 abolishes melanin formation in C. neoformans. This study identifies a novel signaling pathway in which C. neoformans Ipc1 plays a key role in the activation of Pkc1 through the formation of DAG. Importantly, this pathway is essential for melanin production with implications for the pathogenicity of C. neoformans.

Amino Acid Sequence↗
PubMed2000

Molecular epidemiology of Blastomyces dermatitidis.

The inhalation of conidia of Blastomyces dermatitidis, a fungus found in soil, causes disease in humans and animals. We studied the genetic diversity of this pathogen by extracting DNA yeasts and analyzing them with a polymerase chain reaction (PCR)-based typing system we developed, which used restriction fragment analysis of amplicons from the regions between the rDNA repeats and allowed us to class isolates into 3 major groups. Strains were further differentiated by use of PCR fingerprinting with 3 different primers. Fifty-nine isolates collected over 35 years from 15 regions (United States, India, Africa, Canada) were analyzed. Genotypic groups A, B, and C contained 17, 23, and 19 isolates, which were divided into 5, 15, and 12 types, respectively. All 16 isolates from North America in group A were from the upper midwestern United States or Canada, whereas 0 of 20 isolates from the southeastern United States were in group A. Studies of the largest collection from 1 locale (Eagle River, WI), revealed that the soil isolates studied were not responsible for the majority of cases in this outbreak, as previously proposed, and that >1 strain was present in the environment and in patients. Overall, these results provide a tool for the epidemiological study of blastomycosis and illuminate the genetic and geographic diversity of this important pathogen.

Africa↗
PubMed2006

Disseminated blastomycosis in an infant.

Blastomycosis is an uncommon life-threatening disease caused by the dimorphic fungus Blastomyces dermatitidis. Estimates of the percentage of cases occurring in children range from 2% to 11%, and it is exceedingly rare in individuals less than 1 year of age. We present the case of an otherwise healthy infant with disseminated blastomycosis acquired in Northern Ontario and a brief review of the pediatric literature.

Amphotericin B↗
PubMed2001

Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species.

ASPERGILLUS: fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-alpha- and 2,6-alpha-linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R(F) as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6.7 x 10(5) sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.

Aspergillus↗
PubMed1980

Infection of the finger-nail by Pyrenochaeta unguis-hominis.

Infection of the finger-nail of an elderly woman by Pyrenochaeta unguis-hominis is reported. The small lesion was extending slowly into the healthy nail plate. The fungus has been isolated from diseased toe-nails on two previous occasions but its extra-human habitat is unknown.

Aged↗
PubMed2004

B and T cell responses elicited by monoclonal anti-idiotypic antibody (Ab2beta) mimicking gp43 from Paracoccidioides brasiliensis.

Paracoccidioidomycosis is a systemic mycosis endemic in Latin America, with a high prevalence in Brazil, Argentina, Colombia and Venezuela. The aetiological agent of disease is the thermal dimorphic fungus, Paracoccidioides brasiliensis. A glycoprotein of 43 kD (gp43) is the major antigen of P. brasiliensis. Antibodies directed to this antigen are detected in the sera of all patients with paracoccidioidomycosis (PCM). Recently, it has been shown that mice immunized with anti-gp43 monoclonal antibodies (MAbs) (Ab1), induce the idiotypic cascade in the gp43 system, which produced both, anti-Id antibodies (Ab2) and anti-anti-Id antibodies (Ab3). To further characterize the idiotypic cascade modulation in mice immunized with anti-gp43 MAb 17c, hybridomas were produced. Ab2 MAbs named 7.B12 inhibited (>95%) the binding of gp43 to MAb 17c (Ab1), suggesting that this anti-Id MAb bind to the idiotope, thus fulfilling the internal image criteria. To elucidate whether Ab2 MAb could act as antigen in serological assays, instead of gp43, sera from PCM patients were tested. Using an ELISA test, it was observed that antibodies from patients and not normal serum bound to Ab2. However, the ELISA test using Ab2 bound to the solid phase made possible to serologically monitor the patients after antifungal therapy, showing an equivalent curve when compared with ELISA test employing purified gp43. Our results also showed that, when mice were immunized with Ab2beta and their cells were exposed to gp43 in vitro, a T cell proliferation response was observed.

Animals↗
PubMed1994

Polyhydroxynaphthalene reductase involved in melanin biosynthesis in Magnaporthe grisea. Purification, cDNA cloning and sequencing.

During the biosynthesis of fungal melanin, tetrahydroxynaphthalene reductase catalyzes the NADPH-dependent reduction of 1,3,6,8-tetrahydroxynaphthalene (T4HN) into (+)-scytalone and 1,3,8-trihydroxynaphthalene into (-)-vermelone. The enzyme from Magnaporthe grisea, the fungus responsible for rice blast disease, has been purified to homogeneity. It is a tetramer of four identical 30-kDa subunits. A full-length cDNA clone of about 1 kb encoding T4HN reductase has been isolated from a cDNA library constructed in the lambda ZAP II vector and characterized. The clone contains a 846-bp open reading frame. Translation of the DNA sequence gave a 282-residue amino acid sequence with a calculated molecular mass of 29.9 kDa. Sequences corresponding to the amino-terminal part and three internal proteolytic peptides were present in the translated sequence. T4HN reductase exhibits characteristics of the short-chain alcohol dehydrogenase family. The reductase shares 56% identity with a putative ketoreductase involved in aflatoxin biosynthesis in Aspergillus parasiticus.

Amino Acid Sequence↗