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.

Galleria mellonella has been widely used as a heterologous host for fungal pathogens.Melanin has been linked to Fonsecaea monophora virulence.while its role and mechanisms remains unclear.Herein we address this question in an Galleria mellonella infection model using albino and melanin strains of Fonsecaea monophora . Restoration Melanin of Fonseace monophora reduced Its Pathogenicity to Galleria mellonella

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.

Kingdom Protista contains a large group of eukaryotic organisms with diverse lifestyles. We developed the Protist Secretome and Subcellular Proteome Knowledgebase (ProtSecKB) to host information of curated and predicted subcellular locations of all protist proteins. The protist protein sequences were retrieved from UniProtKB, consisting of 1.97 million entries generated from 7,024 species with 101 species including 127 organisms having complete proteomes. The protein subcellular locations were based on curated information and predictions using a set of well evaluated computational tools. The database can be searched using several different types of identifiers, gene names or keyword(s). Secretomes and other subcellular proteomes can be searched or downloaded. BLAST searching against the complete set of protist proteins or secretomes is available. Protein family analysis of secretomes from representing protist species, including Dictyostelium discoideum , Phytophthora infestans , and Trypanosoma cruzi , showed that species with different lifestyles had drastic differences of protein families in their secretomes, which may determine their lifestyles. The database provides an important resource for the protist and biomedical research community. The database is available at http://bioinformatics.ysu.edu/secretomes/protist/index.php.

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.

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.

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

FunSecKB2 is an improved and updated version of the fungal secretome and subcellular proteome, i. e. protein subcellular location, knowledgebase. The fungal protein sequence data were retrieved from UniProtKB, consisting of nearly 2 million entries with 167 species having a complete proteome. The assignments of protein subcellular locations were based on curated information and prediction using seven computational tools. The tools used for subcellular location prediction include SignalP, WoLF PSORT, Phobius, TargetP, TMHMM, FragAnchor, and PS-Scan. Secreted proteins, i.e. secretomes, along with 15 other subcellular proteomes were predicted. The database can be searched by users using several different types of identifiers, gene name or keyword(s). A subcellular proteome from a species can be searched or downloaded. BLAST searching whole fungal protein data or secretomes is available. Community annotation of subcelluar locations based on experimental evidence is also supported. A primary analysis revealed that the secretome size of a fungal species is one of the determining factors to its lifestyle. The Gene Ontology and protein domain analysis of fungal secretomes revealed that fungal secretomes contain a large number of hydrolases, peptidases, oxidoreductases, and lysases, which may have potential applications in bio-processing of chemical wastes or biofuel production. The database provides an important and rich resource for the fungal community looking for protein subcellular location information and performing comparative subcellular proteome analysis. Database URL: http://proteomics.ysu.edu/secretomes/fungi2/index.php

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.

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.

The medically significant yeasts comprise more genera than the traditionally held pathogens in the genera Candida and Cryptococcus. Clinically important yeast forms can also be found among the endemic dimorphic fungi, the dematiaceous molds, and even a noncultureable fungus. Collectively, these fungal pathogens exhibit either teleomorphs of the ascomycota or basidiomycota, or their anamorphic forms express morphological and genetic features consistent with one of these taxa. They comprise a diverse spectrum of fungi that have found the means, primarily by exploiting a host organism's weakened immune system, to become noteworthy mycotic agents. Furthermore, they portend the potential for the spectrum of fungal pathogens to become wider and deeper in the number of etiological agents derived from normally benign species. Vigilance by the general as well as the medical mycologist must be maintained to better understand how to address the future challenges likely to be posed to public health by fungi, particularly the yeasts.

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.

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.

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.