Increased virulence of albino mutant of Fonsecaea monophora in Galleria mellonella.

Fonsecaea monophora has been the predominant pathogen of chromoblastomycosis in Southern China, but its pathogenic mechanism remains unclear. New models are needed to study this infection. In the current study, we examined the role of melanin on the pathogenicity of F. monophora in Galleria mellonella model using melanin and albino strain. Interestingly, the albino mutant strain displayed higher pathogenicity compared to the melanin stain and restoration of melanin of albino mutant could reverse the pathogenicity. Histopathology showed that inflammatory nodules were bigger than that infected with albino cells, which suggested that melanized cells could trigger a robust cellular immune response of G. mellonella than albino cells. The activated immune response in G. mellonella induced by melanized cells might explain the decreased virulence of melanized cells in larvae model. While further study was needed to gain full insights into the molecular immunological mechanism in G. mellonella activated by melanin.

Role of the rttA gene in morphogenesis, stress response, and virulence in the human pathogenic fungus Penicillium marneffei.

Penicillium marneffei is a human pathogenic fungus and the only thermally dimorphic species of the genus. At 25°C, P. marneffei grows as a mycelium that produces conidia in chains. However, when incubated at 37°C or following infection of host tissue, the fungus develops as a fission yeast. Previously, a mutant (strain I133) defective in morphogenesis was generated via Agrobacterium-mediated transformation. Specifically, the rtt109 gene (subsequently designated rttA) in this mutant was interrupted by T-DNA insertion. We characterized strain I133 and the possible roles of the mutated rttA gene in altered P. marneffei phenotypes. At 25°C, the rttA mutant produces fewer conidia than the wild type and a complemented mutant strain, as well as slower rates of conidial germination; however, strain I133 continued to grow as a yeast in 37°C-incubated cultures. Furthermore, whereas the wild type exhibited increased expression of rttA at 37°C in response to the DNA-damaging agent methyl methane sulfonate, strain I133 was hypersensitive to this and other genotoxic agents. Under similar conditions, the rttA mutant exhibited decreased expression of genes associated with carbohydrate metabolism and oxidative stress. Importantly, when compared with the wild-type and the complemented strain, I133 was significantly less virulent in a Galleria infection model when the larvae were incubated at 37°C. Moreover, the mutant exhibited inappropriate phase transition in vivo. In conclusion, the rttA gene plays important roles in morphogenesis, carbohydrate metabolism, stress response, and pathogenesis in P. marneffei, suggesting that this gene may be a potential target for the development of antifungal compounds.

Role of the yakA gene in morphogenesis and stress response in Penicillium marneffei.

Penicillium marneffei is a thermally dimorphic fungus and a highly significant pathogen of immunocompromised individuals living in or having travelled in south-east Asia. At 25 °C, P. marneffei grows filamentously. Under the appropriate conditions, these filaments (hyphae) produce conidiophores bearing chains of conidia. Yet, when incubated at 37 °C, or upon infecting host tissue, P. marneffei grows as a yeast that divides by binary fission. Previously, an Agrobacterium-mediated transformation system was used to randomly mutagenize P. marneffei, resulting in the isolation of a mutant defective in normal patterns of morphogenesis and conidiogenesis. The interrupted gene was identified as yakA. In the current study, we demonstrate that the yakA mutant produced fewer conidia at 25 °C than the wild-type and a complemented strain. In addition, disruption of the yakA gene resulted in early conidial germination and perturbation of cell wall integrity. The yakA mutant exhibited abnormal chitin distribution while growing at 25 °C, but not at 37 °C. Interestingly, at both temperatures, the yakA mutant possessed increased chitin content, which was accompanied by amplified transcription of two chitin synthase genes, chsB and chsG. Moreover, the expression of yakA was induced during post-exponential-phase growth as well as by heat shock. Thus, yakA is required for normal patterns of development, cell wall integrity, chitin deposition, appropriate chs expression and heat stress response in P. marneffei.

Spermidine is required for morphogenesis in the human pathogenic fungus, Penicillium marneffei.

Penicillium marneffei is a thermally dimorphic fungus that is a highly significant pathogen of immune compromised persons living or having traveled in Southeast Asia. When cultured at 25°C, the wild-type strain of P. marneffei exhibits a mycelial morphology that is marked by the development of specialized structures bearing conidia. Incubation of the wild type at 37°C, however, promotes the development of a yeast form that divides by fission. Development of the yeast morphology in vivo appears to be requisite for pathogenesis. In a prior study using Agrobacterium-mediated transformation for random mutagenesis via T-DNA integration, we generated a morphological mutant (strain I6) defective in conidiation. The T-DNA insertion site in strain I6 was determined to be within the gene encoding S-adenosylmethionine decarboxylase (sadA), an enzyme critical to spermidine biosynthesis. In the present study, we demonstrated that strain I6 was able to grow on rich media in either the mold or yeast forms at 25°C and 37°C, respectively. However, reduced growth of strain I6 was observed on minimal medium at either temperature. In addition, strain I6 produced mycelia with impaired conidiation on minimal medium at 25°C. Supplementation of minimal medium with spermidine restored the ability of strain I6 to produce conidia at 25°C and promoted yeast development at 37°C. Moreover, conidia of strain I6 exhibited poor germination frequencies in the absence of this polyamine. All three of these processes (conidiogenesis, germination, and growth) were reinstated in strain I6 by complementation of the partially deleted of sadA gene by ectopic insertion of an intact wild-type copy. These results augment prior observations that spermidine biosynthesis is essential to normal growth, conidiogenesis, spore germination, and dimorphism in a variety of fungi. Given the presumption that P. marneffei infections are initiated following inhalation of conidia, and that pathogenesis is dependent upon yeast development, this study further suggests that the spermidine biosynthetic pathway may serve as a potential target for combating infections by this medically important fungus.

Penicillium marneffei actin expression during phase transition, oxidative stress, and macrophage infection.

Penicillium marneffei is an opportunistic fungal pathogen that exhibits thermally regulated dimorphism. At 25°C, this fungus grows vegetatively as mycelia, but at 37°C or upon invasion of a host, a fission yeast form is established. Yet, despite increased numbers of molecular studies involving this fungus, the role of P. marneffei stress response-related proteins is not well characterized. Actin is one of the proteins that have been proposed to play a role not only in cell transition, but also in thermo-adaptation. Here, we report the isolation and characterization of the actin encoding gene, actA, from P. marneffei. Examination of the deduced amino acid sequence of the ActA protein revealed that it is closely related to Aspergillus nidulans and Aspergillus clavatus. Northern blot analysis of actin expression during the mycelium to yeast phase transition of P. marneffei showed that the actA transcripts were initially upregulated soon after shifting the incubation temperature from 25°C to 37°C, but subsequently decreased slightly and did not change during further growth or under stress conditions. When cultures were started with conidia, upregulation of actA gene was found to correlate with germ tube production at either 25°C or 37°C. However, the relative expression level of actA transcripts again showed no significant differences in different cell types (conidia, mycelium, and yeast cells) or during macrophage infection. These results suggest that actin may play an important role in the early stages of cellular development, but not in environmental stress responses.

An improved Agrobacterium-mediated transformation system for the functional genetic analysis of Penicillium marneffei.

We have developed an improved Agrobacterium-mediated transformation (AMT) system for the functional genetic analysis of Penicillium marneffei, a thermally dimorphic, human pathogenic fungus. Our AMT protocol included the use of conidia or pre-germinated conidia of P. marneffei as the host recipient for T-DNA from Agrobacterium tumefaciens and co-cultivation at 28°C for 36 hours. Bleomycin-resistant transformants were selected as yeast-like colonies following incubation at 37°C. The efficiency of transformation was approximately 123 ± 3.27 and 239 ± 13.12 transformants per plate when using 5 × 10(4) conidia and pre-germinated conidia as starting materials, respectively. Southern blot analysis demonstrated that 95% of transformants contained single copies of T-DNA. Inverse PCR was employed for identifying flanking sequences at the T-DNA insertion sites. Analysis of these sequences indicated that integration occurred as random recombination events. Among the mutants isolated were previously described stuA and gasC defective strains. These AMT-derived mutants possessed single T-DNA integrations within their particular coding sequences. In addition, other morphological and pigmentation mutants possessing a variety of gene-specific defects were isolated, including two mutants having T-DNA integrations within putative promoter regions. One of the latter integration events was accompanied by the deletion of the entire corresponding gene. Collectively, these results indicated that AMT could be used for large-scale, functional genetic analyses in P. marneffei. Such analyses can potentially facilitate the identification of those genetic elements related to morphogenesis, as well as pathogenesis in this medically important fungus.

Karyotype determination and gene mapping in two clinical isolates of Penicillium marneffei.

Electrophoretic karyotypes from two clinical isolates of Penicillium marneffei were derived using contoured-clamped homogeneous electric field (CHEF) gel electrophoresis. Five chromosome-sized DNA fragments were resolved from each isolate. Chromosomal length polymorphisms were evident among the three largest molecules. Based upon these electrophoretic separation patterns, individual chromosomes were estimated to range in size from 2.0 to > or = 6.3 Mbp with a total genome size of 20.6 to > or = 21.6 Mbp. However, Southern blot analysis using a telomeric probe suggests that P. marneffei may possess as many as seven chromosomes, including two pairs of molecules that co-migrate under the CHEF separation conditions employed in this study. Hence, the total genome size of P. marneffei may be as large as 25.7 to > or = 26.7 Mbp. Further hybridization analysis mapped four chitin synthase homologues to specific chromosomes as well as genes for chitinase, malate synthase, isocitrate lyase, isocitrate dehydrogenase, 5.8S rRNA, and 23S rRNA. The mapping analyses also suggested the existence of multiple chitinase gene homologues in P. marneffei. The collective results of this investigation provide additional foundations for facilitating the genetic characterization of P. marneffei and the molecular epidemiology of penicilliosis due to this fungus.

Isolation and expression of heat shock protein 30 gene from Penicillium marneffei.

Penicillium marneffei is a dimorphic fungus that can cause disseminated mycosis, especially in AIDS patients. The role of heat shock proteins and stress response-related proteins in P. marneffei remains unknown. In this study, we isolated a cDNA encoding for heat shock protein 30 (Hsp30) of P. marneffei using an antibody screening method. The DNA sequence and deduced amino acid sequence analysis showed high homology to other fungal hsp30 genes. Expression of P. marneffei hsp30 in response to temperature increase was determined by Northern blot analysis. A high level of hsp30 transcript was detected in yeast cells grown at 37 degrees C, whereas a very low or undetectable transcript level was observed in mycelial cells at 25 degrees C. A recombinant Hsp30 protein was produced and tested preliminarily for its immunoreactivity with sera from P. marneffei-infected AIDS patients using Western blot analysis. The positive immunoblot result, with some serum samples, confirmed the antigenic property of the Hsp30. Collectively, the high response of hsp30 to temperature increase could indicate it may play a role in heat stress response and cell adaptation. This is the first report showing that this small heat shock protein could elicit the human immune response.

Molecular cloning and characterization of WdTUP1, a gene that encodes a potential transcriptional repressor important for yeast-hyphal transitions in Wangiella (Exophiala) dermatitidis.

The general transcriptional repressor Tup1p is known to influence cell development in many fungi. To determine whether the Tup1p ortholog (WdTup1p) of Wangiella dermatitidis also influences cellular development in this melanized, polymorphic human pathogen, the gene (WdTUP1) that encodes this transcription factor was isolated, sequenced and disrupted. Phylogenetic analysis showed that the WdTup1p sequence was closely related to homologues in other polymorphic, conidiogenous fungi. Disruption of WdTUP1 produced mutants (wdtup1Delta) with pronounced growth and cellular abnormalities, including slow growth on various agar media and exclusively as a filamentous morphotype in liquid media. We concluded that WdTup1p represents an important switch regulator that controls the yeast-to-filamentous growth transition. However, detailed observations of the filamentous growth of the disruption mutant showed that the hyphae produced by the wdtup1Delta mutants, unlike those of the wild-type, were arrested at a stage prior to the formation of true hyphae and subsequent conidia production.

Protein profiling of the dimorphic, pathogenic fungus, Penicillium marneffei.

BACKGROUND: Penicillium marneffei is a pathogenic fungus that afflicts immunocompromised individuals having lived or traveled in Southeast Asia. This species is unique in that it is the only dimorphic member of the genus. Dimorphism results from a process, termed phase transition, which is regulated by temperature of incubation. At room temperature, the fungus grows filamentously (mould phase), but at body temperature (37 degrees C), a uninucleate yeast form develops that reproduces by fission. Formation of the yeast phase appears to be a requisite for pathogenicity. To date, no genes have been identified in P. marneffei that strictly induce mould-to-yeast phase conversion. In an effort to help identify potential gene products associated with morphogenesis, protein profiles were generated from the yeast and mould phases of P. marneffei. RESULTS: Whole cell proteins from the early stages of mould and yeast development in P. marneffei were resolved by two-dimensional gel electrophoresis. Selected proteins were recovered and sequenced by capillary-liquid chromatography-nanospray tandem mass spectrometry. Putative identifications were derived by searching available databases for homologous fungal sequences. Proteins found common to both mould and yeast phases included the signal transduction proteins cyclophilin and a RACK1-like ortholog, as well as those related to general metabolism, energy production, and protection from oxygen radicals. Many of the mould-specific proteins identified possessed similar functions. By comparison, proteins exhibiting increased expression during development of the parasitic yeast phase comprised those involved in heat-shock responses, general metabolism, and cell-wall biosynthesis, as well as a small GTPase that regulates nuclear membrane transport and mitotic processes in fungi. The cognate gene encoding the latter protein, designated RanA, was subsequently cloned and characterized. The P. marneffei RanA protein sequence, which contained the signature motif of Ran-GTPases, exhibited 90% homology to homologous Aspergillus proteins. CONCLUSION: This study clearly demonstrates the utility of proteomic approaches to studying dimorphism in P. marneffei. Moreover, this strategy complements and extends current genetic methodologies directed towards understanding the molecular mechanisms of phase transition. Finally, the documented increased levels of RanA expression suggest that cellular development in this fungus involves additional signaling mechanisms than have been previously described in P. marneffei.

Molecular analysis of the Penicillium marneffei glyceraldehyde-3-phosphate dehydrogenase-encoding gene (gpdA) and differential expression of gpdA and the isocitrate lyase-encoding gene (acuD) upon internalization by murine macrophages.

Penicillium marneffei is an intracellular dimorphic fungus that can cause a fatal disseminated disease in human immunodeficiency virus-infected patients. The factors that affect the pathogenicity of this fungus remain unclear. Here, we report the isolation and characterization of the gpdA cDNA and genomic clones encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in P. marneffei. Phylogenetic analysis of GAPDH amino acid sequences demonstrated the evolutionary relationship of P. marneffei to other fungi, including the intracellular pathogen Ajellomyces capsulatus. To assess the central importance of phagocytic cells in defence against P. marneffei infection, we used Northern blotting to investigate the response of the isocitrate lyase-encoding gene (acuD) and gpdA to nutrient deprivation inside macrophages. The results revealed that after macrophage internalization, the gene involved in the glyoxylate cycle, acuD, showed higher expression levels as early as 2 h from the start of co-incubation, and the differential expression could be observed again at 8 h after infection. In contrast, the expression of gpdA was downregulated in the yeast phase, as well as during macrophage infection after 2, 4 and 8 h of infection. The induction of P. marneffei acuD was shown to be coordinated with the downregulation of the glycolytic gpdA gene, implying that the cytoplasmic environment of macrophages is deficient in glucose and the glyoxylate pathway could be used by this pathogen to allow subsistence on two-carbon compounds within the host cell following its intracellular persistence.

Adaptation to macrophage killing by Talaromyces marneffei.

Talaromyces (Penicillium) marneffei is an important opportunistic fungal pathogen. It causes disseminated infection in immunocompromised patients especially in Southeast Asian countries. The pathogenicity of T. marneffei depends on the ability of the fungus to survive the killing process and replicate inside the macrophage. Major stresses inside the phagosome of macrophages are heat, oxidative substances and nutrient deprivation. The coping strategies of this pathogen with these stresses are under investigation. This paper summarizes factors relating to the stress responses that contribute to the intracellular survival of T. marneffei. These include molecules in the MAP signal transduction cascade, heat shock proteins, antioxidant enzymes and enzymes responsible in nutrient retrieval. There is speculation that the ability of T. marneffei to withstand these defenses plays an important role in its pathogenicity.

The yapA Encodes bZIP Transcription Factor Involved in Stress Tolerance in Pathogenic Fungus Talaromyces marneffei.

Talaromyces marneffei, formerly Penicillium marneffei, is a thermally dimorphic fungus. It causes a fatal disseminated disease in patients infected with the human immunodeficiency virus (HIV). Studies on the stress defense mechanism of T. marneffei can lead to a better understanding of the pathogenicity and the progression of the disease due to this fungus. The basic leucine-zipper (bZip) transcription factor gene in Saccharomyces cerevisiae, named yap1 (yeast activating protein-1), is known as a crucial central regulator of stress responses including those caused by oxidative agents, cadmium, and drugs. An ortholog of yap1, designated yapA, was identified in T. marneffei. We found that the yapA gene was involved in growth and fungal cell development. The yapA deletion mutant exhibited delays in the rate of growth, germination, and conidiation. Surprisingly, the yapA gene was also involved in the pigmentation of T. marneffei. Moreover, the mutant was sensitive to oxidative stressors such as H2O2 and menadione, similar to S. cerevisiae yap1 mutant, as well as the nitrosative stressor NaNO2. In addition, the yapA mutant demonstrated significantly decreased survival in human macrophage THP-1 compared to wild-type and complemented strains. This study reveals the role of yapA in fungal growth, cell development, stress response, and potential virulence in T. marneffei.

MetazSecKB: the human and animal secretome and subcellular proteome knowledgebase.

The subcellular location of a protein is a key factor in determining the molecular function of the protein in an organism. MetazSecKB is a secretome and subcellular proteome knowledgebase specifically designed for metazoan, i.e. human and animals. The protein sequence data, consisting of over 4 million entries with 121 species having a complete proteome, were retrieved from UniProtKB. Protein subcellular locations including secreted and 15 other subcellular locations were assigned based on either curated experimental evidence or prediction using seven computational tools. The protein or subcellular proteome data can be searched and downloaded using several different types of identifiers, gene name or keyword(s), and species. BLAST search and community annotation of subcellular locations are also supported. Our primary analysis revealed that the proteome sizes, secretome sizes and other subcellular proteome sizes vary tremendously in different animal species. The proportions of secretomes vary from 3 to 22% (average 8%) in metazoa species. The proportions of other major subcellular proteomes ranged approximately 21-43% (average 31%) in cytoplasm, 20-37% (average 30%) in nucleus, 3-19% (average 12%) as plasma membrane proteins and 3-9% (average 6%) in mitochondria. We also compared the protein families in secretomes of different primates. The Gene Ontology and protein family domain analysis of human secreted proteins revealed that these proteins play important roles in regulation of human structure development, signal transduction, immune systems and many other biological processes. Database URL: http://proteomics.ysu.edu/secretomes/animal/index.php.

Capillary electrophoresis for protein profiling of the dimorphic, Pathogenic Fungus, Penicillium marneffei.

Penicillium marneffei is an endemic, dimorphic fungus that exhibits very significant morbidity among immune compromised persons living or having traveled in Southeast Asia. The dimorphic nature of P. marneffei, which is believed to be a major contributing factor to infection by this fungus, is thermally regulated. At 25 °C, the fungus grows as a mold, but converts to a yeast phase when incubated at 37 °C. Hence, protein profiling of these developing forms will help ascertain the underpinning molecular mechanisms associated with this phase transition, and perhaps provide clues to virulence in this pathogenic fungus. This chapter outlines the basic procedures previously used to demonstrate distinct differences in protein expression between the mold and yeast phases of P. marneffei.

Insights into the pathogenicity of Penicillium marneffei.

Penicillium marneffei is a significant pathogen of AIDS patients in Southeast Asia. This fungus is unique in that it is the only dimorphic member of the genus. Pathogenesis of P. marneffei requires the saprobic mold form to undergo a morphological change upon tissue invasion. The in vivo form of this fungus reproduces as a fission yeast that capably evades the host immune system. The processes that control these morphological changes, better termed as phase transition, can be replicated in vitro by incubation of the mold form at 37 degrees C. The unidentified molecular mechanisms regulating phase transition in this fungus are now being uncovered using modern methodologies and novel strategies. A better comprehension of these underlying regulatory pathways will provide insight into eukaryotic cellular development as well as the potential factors responsible for infections caused by P. marneffei and other fungi. Such knowledge may lead to better chemotherapeutic interventions of fungal diseases.

The copper, zinc superoxide dismutase gene of Penicillium marneffei: cloning, characterization, and differential expression during phase transition and macrophage infection.

Superoxide dismutase (SOD) is an enzyme that converts superoxide radicals into hydrogen peroxide and oxygen molecules. SOD has been shown to contribute to the virulence of many human-pathogenic fungi through its ability to neutralize toxic levels of reactive oxygen species generated by the host. SOD has also been speculated to be important in the pathogenesis of fungal infections, but the role of this enzyme has not been rigorously investigated. In this report, we isolated and characterized the copper, zinc superoxide dismutase gene, designated sodA, from the important human pathogenic fungus, Penicillium marneffei. The putative SodA polypeptide consisted of 154 amino acids and exhibited a significant level of similarity to other fungal Cu, Zn SODs. Differential expression of the sodA gene in P. marneffei was demonstrated by semi-quantitative RT-PCR. Apparently, the sodA transcript accumulated in conidia, but expression was downregulated in the mycelia phase. In contrast, transcript expression was upregulated in the yeast phase as well as during macrophage infection. The significantly higher expression of the sodA transcript during macrophage infection suggests that this gene might play an important role in stress responses and in the adaptation of P. marneffei to the internal macrophage environment. The latter may serve as a putative virulence factor of this fungus allowing for survival in the host cell.

Source tracking of Escherichia coli by 16S-23S intergenic spacer region denaturing gradient gel electrophoresis (DGGE) of the rrnB ribosomal operon.

This research validates a novel approach for source tracking based on denaturing gradient gel electrophoresis (DGGE) analysis of DNA extracted from Escherichia coli isolates. Escherichia coli from different animal sources and from river samples upstream from, at, and downstream of a combined sewer overflow were subjected to DGGE to determine sequence variations within the 16S-23S intergenic spacer region (ISR) of the rrnB ribosomal operon. The ISR was analyzed to determine if E. coli isolates from various animal sources could be differentiated from each other. DNA isolated from the E. coli animal sources was PCR amplified to isolate the rrnB operon. To prevent amplification of all 7 E. coli ribosomal operons by PCR amplification using universal primers, sequence-specific primers were utilized for the rrnB operon. Another primer set was then used to prepare samples of the 16S-23S ISR for DGGE. Comparison of PCR-DGGE results between human and animal sources revealed differences in the distribution and frequency of the DGGE bands produced. Human and Canada Goose isolates had the most unique distribution patterns and the highest percent of unique isolates and were grouped separately from all other animal sources. Method validation suggests that there are enough host specificity and genetic differences for use in the field. Field results at and around a combined sewer overflow indicate that this method can be used for microbial source tracking.

Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects.

Penicillium marneffei infection is an important emerging public health problem, especially among patients infected with human immunodeficiency virus in the areas of endemicity in southeast Asia, India, and China. Within these regions, P. marneffei infection is regarded as an AIDS-defining illness, and the severity of the disease depends on the immunological status of the infected individual. Early diagnosis by serologic and molecular assay-based methods have been developed and are proving to be important in diagnosing infection. The occurrence of natural reservoirs and the molecular epidemiology of P. marneffei have been studied; however, the natural history and mode of transmission of the organism remain unclear. Soil exposure, especially during the rainy season, has been suggested to be a critical risk factor. Using a highly discriminatory molecular technique, multilocus microsatellite typing, to characterize this fungus, several isolates from bamboo rats and humans were shown to share identical multilocus genotypes. These data suggest either that transmission of P. marneffei may occur from rodents to humans or that rodents and humans are coinfected from common environmental sources. These putative natural cycles of P. marneffei infection need further investigation. Studies on the fungal genetics of P. marneffei have been focused on the characterization of genetic determinants that may play important roles in asexual development, mycelial-to-yeast phase transition, and the expression of antigenic determinants. Molecular studies have identified several genes involved in germination, hyphal development, conidiogenesis, and yeast cell polarity. A number of functionally important genes, such as the malate synthase- and catalase-peroxidase protein-encoding genes, have been identified as being upregulated in the yeast phase. Future investigations pertaining to the roles of these genes in host-fungus interactions may provide the key knowledge to understanding the pathogenicity of P. marneffei.