Rapid and robust identification of clinical isolates of Talaromyces marneffei based on MALDI-TOF mass spectrometry or dimorphism in Galleria mellonella.

Talaromyces marneffei is a thermally dimorphic fungal pathogen that causes serious infections particularly in patients with human immunodeficiency virus (HIV). Although the mould form typically produces a characteristic red-diffusing pigment, and conidia from penicillate heads, several nonpathogenic Talaromyces/Penicillium species are morphologically and phenotypically similar. While those other species do not exhibit thermal dimorphism, conversion of T. marneffei to the distinctive fission yeast form in vitro is arduous and frequently incomplete. Here we show that T. marneffei can be rapidly and unambiguously discriminated from related nonpathogenic Talaromyces/Penicillium spp., either by matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) mass spectrometry or conversion to fission yeast after introduction into Galleria mellonella. Conversion of T. marneffei conidia to the fission yeast form in G. mellonella larvae occurred as early as 24 h post inoculation at 37oC. Identification by MALDI-TOF was possible after supplementation of the commercial Bruker database with in-house mass spectral profiles created from either the yeast or mycelial phase of T. marneffei. In addition, we show that in-house generated mass spectral profiles could be successfully used to identify T. marneffei with a recently published on-line MALDI-TOF database, circumventing the need to create extensive in-house additional databases for rarely encountered fungal pathogens.

Sixty Years from Segretain's Description: What Have We Learned and Should Learn About the Basic Mycology of Talaromyces marneffei?

The fungus Talaromyces marneffei was described by Professor Gabriel Segretain in 1959, originally as a member of the genus Penicillium. As early as 60 years ago, its peculiarity in exhibiting temperature-dependent morphological dimorphism, its characteristic ability to secrete diffusing red pigment during the mycelial phase and its pathogenicity have already been recognised. Six decades have passed, and our understanding on this intriguing fungus has improved. Apart from the clinical aspect, we have gained a glimpse on its taxonomy, animal or environmental source(s), mechanism of thermal dimorphism, molecular genetics, virulence as well as pathogenesis. However, we are still on our way to get out of the talaromycosis mist. A lot more collective endeavour on T. marneffei research is needed to solve the jigsaw puzzle.

Differentially regulated high-affinity iron assimilation systems support growth of the various cell types in the dimorphic pathogen Talaromyces marneffei.

Iron is a key trace element important for many biochemical processes and its availability varies with the environment. For human pathogenic fungi iron acquisition can be particularly problematical because host cells sequester free iron as part of the acute-phase response to infection. Fungi rely on high-affinity iron uptake systems, such as reductive iron assimilation (RIA) and siderophore-mediated iron uptake (non-RIA). These have been extensively studied in pathogenic fungi that exist outside of host cells, but much less is known for intracellular fungal pathogens. Talaromyces marneffei is a dimorphic fungal pathogen endemic to Southeast Asia. In the host T. marneffei resides within macrophages where it grows as a fission yeast. T. marneffei has genes of both iron assimilation systems as well as a paralogue of the siderophore biosynthetic gene sidA, designated sidX. Unlike other fungi, deletion of sidA or sidX resulted in cell type-specific effects. Mutant analysis showed that T. marneffei yeast cells also employ RIA for iron acquisition, providing an additional system in this cell type that differs substantially from hyphal cells. These data illustrate the specialized iron acquisition systems used by the different cell types of a dimorphic fungal pathogen and highlight the complexity in siderophore-biosynthetic pathways amongst fungi.

Talaromyces rubrifaciens, a new species discovered from heating, ventilation and air conditioning systems in China.

A new Talaromyces species, T. rubrifaciens, was isolated from supply air outlets of heating, ventilation and air conditioning (HVAC) systems in three kinds of public building in Beijing and Nanjing, China. Morphologically it exhibits many characters of section Trachyspermi but is distinguished from other species of this section by restricted growth and broad and strictly biverticillate conidiophores. Phylogenetic analyses based on the internal transcribed spacer rDNA (ITS), ß-tubulin (BenA), calmodulin (CaM) and RNA polymerase second largest subunit (RPB2) genes reveal that T. rubrifaciens is a distinct species in section Trachyspermi.

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.

Cloning, heterologous expression, and characterization of the xylitol and L-arabitol dehydrogenase genes, Texdh and Telad, from the thermophilic fungus Talaromyces emersonii.

The genes encoding xylitol dehydrogenase (Texdh) and L: -arabitol dehydrogenase (Telad) are involved in the fungal pentose pathway and were isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli, and the products purified to homogeneity. TeXDH showed activity toward xylitol and D: -sorbitol. TeLAD was active with L: -arabitol, xylitol, and D: -sorbitol. Phylogenetic analysis showed TeLAD has evolved from D: -sorbitol dehydrogenase as a result of environmental adaptation. Substrate specificity studies indicate that TeXDH is likely to have evolved from the more broadly acting TeLAD. Texdh and Telad expression was inducible by the same carbon sources responsible for induction of genes involved in biomass degradation, suggesting for the first time a coordinated regulatory control mechanism for expression of genes encoding extracellular hydrolases and intracellular metabolic genes in the pentose utilization pathways of T. emersonii. These data also suggest that TeXDH and TeLAD may be valuable in the production of xylitol, L: -arabitol, and ethanol from renewable resources rich in pentose sugars.

Talaromyces indigoticus Takada & Udagawa, the first record for Panama and the American continent.

In a survey of mycotic human skin and nail lesions in Chiriquí, Western Panama, Talaromyces indigoticus was isolated in one case. This ascomycete is characterised by formation of gymnothecia containing oval, spinose and yellow ascospores, which become blue in mounting fluids, and by short monoverticillate or biverticillate penicilli. It is here reported the first time for Panama and the American continent. Because the strain was isolated from an onychomycosis together with a dermatophyte and other fungal strains, the pathological impact of Talaromyces indigoticus is not evident.

Characterization of a novel yeast phase-specific antigen expressed during in vitro thermal phase transition of Talaromyces marneffei.

Talaromyces marneffei is a dimorphic fungus that has emerged as an opportunistic pathogen particularly in individuals with HIV/AIDS. Since its dimorphism has been associated with its virulence, the transition from mold to yeast-like cells might be important for fungal pathogenesis, including its survival inside of phagocytic host cells. We investigated the expression of yeast antigen of T. marneffei using a yeast-specific monoclonal antibody (MAb) 4D1 during phase transition. We found that MAb 4D1 recognizes and binds to antigenic epitopes on the surface of yeast cells. Antibody to antigenic determinant binding was associated with time of exposure, mold to yeast conversion, and mammalian temperature. We also demonstrated that MAb 4D1 binds to and recognizes conidia to yeast cells' transition inside of a human monocyte-like THP-1 cells line. Our studies are important because we demonstrated that MAb 4D1 can be used as a tool to study T. marneffei virulence, furthering the understanding of the therapeutic potential of passive immunity in this fungal pathogenesis.

Talaromyces pinophilus strain AUN-1 as a novel mycoparasite of Botrytis cinerea, the pathogen of onion scape and umbel blights.

This study aimed to investigate the mycoparasitism of Botrytis cinerea, the pathogen of scape and umbel blights of onion seed crops, by endophytic Talaromyces pinophilus. The dual culture test showed that the antagonistic potentiality of T. pinophilus against B. cinerea depend on the mycoparasitism that was morphologically detected by the formation of mycelial overgrowth. Moreover, the light micrograph of the mycelia at the contact zone exhibited that the hyphae of T. pinophilus penetrated and grew intracellularly inside the hyphae of B. cinerea. A more illustrative figure of the establishment of coiled hyphae as well as the conformation of the penetration process was assayed by SEM and TEM analyses. SEM micrograph revealed that the hyphae of T. pinophilus grew along hyphae of B. cinerea, attached, coiled around the host hypha and generated pseudoappressorium. A clear disintegration of cell wall of the host hypha was observed at the penetration site. The micrographs of TEM exhibited the ability of T. pinophilus to produce pseudoappressorium, penetrate and then entere a hypha of B. cinerea causing distinct cytoplasmic disorganization. High activities of cell wall degrading enzymes (chitinase, lipase and protease) involved in the mycoparasitism were evaluated by the endophytic T. pinophilus. In conclusion, this study demonstrated that the endophytic T. pinophilus may be a promising biocontrol agent against phytopathogenic fungi instead of chemical fungicides.

New species of Talaromyces isolated from maize, indoor air, and other substrates.

Talaromyces strains isolated from maize seeds and the built environment were examined taxonomically because they could not be identified as previously described species. Using phenotypic analysis, DNA sequencing, and phylogenetic and concordance analyses, the authors discovered and described 10 new species in sect. Islandici and 1 new species in sect. Subinflati. Taxonomic novelties in sect. Islandici are Talaromyces delawarensis, T. herodensis, T. juglandicola, T. kilbournensis, T. novojersensis, T. ricevillensis, T. rogersiae, T. siglerae, T. subtropicalis, and T. tiftonensis, and the species from sect. Subinflata is T. tzapotlensis. The isolate of T. siglerae is unusual in Talaromyces because it produced a Sagenomella-like anamorph, but phylogenetic analysis placed it in Talaromyces. Talaromyces rotundus is known from a few isolates, but searches with internal transcribed spacer (ITS) sequences in GenBank revealed that it is commonly endolichenous with Lasallia hispanica. Talaromyces wortmannii also has a role as an endophyte of the aquatic plant Persicaria amphibia, based on ITS sequence records from GenBank.

Extensive Metabolic Remodeling Differentiates Non-pathogenic and Pathogenic Growth Forms of the Dimorphic Pathogen Talaromyces marneffei.

Fungal infections are an increasing public health problem, particularly in immunocompromised individuals. While these pathogenic fungi show polyphyletic origins with closely related non-pathogenic species, many undergo morphological transitions to produce pathogenic cell types that are associated with increased virulence. However, the characteristics of these pathogenic cells that contribute to virulence are poorly defined. Talaromyces marneffei grows as a non-pathogenic hyphal form at 25°C but undergoes a dimorphic transition to a pathogenic yeast form at 37°C in vitro and following inhalation of asexual conidia by a host. Here we show that this transition is associated with major changes in central carbon metabolism, and that these changes are correlated with increased virulence of the yeast form. Comprehensive metabolite profiling and 13C-labeling studies showed that hyphal cells exhibited very active glycolytic metabolism and contain low levels of internal carbohydrate reserves. In contrast, yeast cells fully catabolized glucose in the mitochondrial TCA cycle, and store excess glucose in large intracellular pools of trehalose and mannitol. Inhibition of the yeast TCA cycle inhibited replication in culture and in host cells. Yeast, but not hyphae, were also able to use myo-inositol and amino acids as secondary carbon sources, which may support their survival in host macrophages. These analyses suggest that T. marneffei yeast cells exhibit a more efficient oxidative metabolism and are capable of utilizing a diverse range of carbon sources, which contributes to their virulence in animal tissues, highlighting the importance of dimorphic switching in pathogenic yeast.

Organism-wide studies into pathogenicity and morphogenesis in Talaromyces marneffei.

Organism-wide approaches examining the genetic mechanisms controlling growth and proliferation have proven to be a powerful tool in the study of pathogenic fungi. For many fungal pathogens techniques to study transcription and protein expression are particularly useful, and offer insights into infection processes by these species. Here we discuss the use of approaches such as differential display, suppression subtractive hybridization, microarray, RNA-seq, proteomics, genetic manipulation and infection models for the AIDS-defining pathogen Talaromyces marneffei. Together these methods have broadened our understanding of the biological processes, and genes that underlie them, which are involved in switching between the saprophytic and pathogenic states of T. marneffei, the maintenance of these two specialized cell types and its ability to cause disease.

Physicochemical properties of thermotolerant extracellular ß-glucosidase from Talaromyces thermophilus and enzymatic synthesis of cello-oligosaccharides.

A thermophilic fungus, Talaromyces thermophilus that produces a novel thermotolerant extra-cellular ß-glucosidase (Bgl.tls), was isolated from Tunisian soil samples. The enzyme was purified from the culture filtrates of T. thermophilus grown on lactose using gel filtration, ion exchange chromatography and FPLC. The monomeric enzyme had a molecular mass of 116.0 kDa and a high specific activity of 1429 UI/mg. Bgl.tls exhibited optimal activity at pH 5.0 and 65 °C. It was also stable over a wide range of pH (4.0-10.0) and stable at 50 °C for 34 h. Bgl.tls retained about 80% of its initial activity after 1.0 hours of preincubation at 60 °C. The Km and Vmax values recorded for pNPG were 0.25 mM and 228.7 µmol min(-1), respectively. Bgl.tls was activated by Mn(2+), Mg(2+), Ca(2+) and Co(2+) but obviously inhibited by Fe(2+) and Cu(2+). It was able to hydrolyze a variety of aryl / alkyl -ß-glucosides and disaccharides as well as (1 → 6) and (1 → 4)-ß-glucosidic linkages and α-glycosidic substrates, thus providing evidence for its broad substrate specificity. The enzyme also displayed high hydrolytic and transglycosylation activities. Overall, this study is the first report on the purification and physicochemical properties of a ß-glucosidase secreted by T. thermophilus. The cello-oligosaccharides synthesized by this enzyme within 2 h were mainly cellotriose, cellotetraose and cellopentaose identified by HPLC and ESI-MS techniques.

Purification and characterization of a highly efficient calcium-independent α-amylase from Talaromyces pinophilus 1-95.

Alpha-amylase is a very important enzyme in the starch conversion process. Most of the α-amylases are calcium-dependent and exhibit poor performance in the simultaneous saccharification and fermentation process of industrial bioethanol production that uses starch as feedstock. In this study, an extracellular amylolytic enzyme was purified from the culture broth of newly isolated Talaromyces pinophilus strain 1-95. The purified amylolytic enzyme, with an apparent molecular weight of 58 kDa on SDS-PAGE, hydrolyzed maltopentaose, maltohexaose, and maltoheptaose into mainly maltose and maltotriose and minor amount of glucose, confirming the endo-acting mode of the enzyme, and hence, was named Talaromyces pinophilus α-amylase (TpAA). TpAA was most active at pH 4.0-5.0 (with the temperature held at 37°C) and 55°C (at pH 5.0), and stable within the pH range of 5.0-9.5 (at 4°C) and below 45°C (at pH 5.0). Interestingly, the Ca2+ did not improve its enzymatic activity, optimal temperature, or thermostability of the enzyme, indicating that the TpAA was Ca2+-independent. TpAA displayed higher enzyme activity toward malto-oligosaccharides and dextrin than other previously reported α-amylases. This highly active Ca2+-independent α-amylase may have potential applications in starch-to-ethanol conversion process.

Two novel Talaromyces species isolated from medicinal crops in Korea.

Two novel biverticillate Talaromyces species, T. angelicus and T. cnidii, were collected from the medicinal crops Angelica gigas and Cnidium officinale, respectively, in Korea. Phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region and the ß-tubulin gene as well as morphological analyses revealed that the two species differ from any known Talaromyces species. Talaromyces angelicus is related to T. flavovirens in the phylogeny of the ITS region, but the new species is grouped together with Penicillium liani and T. pinophilus in terms of its ß-tubulin phylogeny, and its growth rate on Czapek yeast autolysate differs from that of T. flavovirens. Talaromyces cnidii is phylogenetically similar to T. siamensis, but exhibits differences in the morphologies of the colony margin, metulae, and conidia.

Granulomatous lymphadenitis caused by Talaromyces helicus in a Labrador Retriever.

A 3-year-old spayed female Labrador Retriever was presented for right prescapular lymphadenomegaly. Examination of fine-needle aspirates and impression smears of the node revealed many short hyphal structures found within macrophages and extracellularly. Hyphae were approximately 3 µm in diameter, were irregularly septate with nonparallel walls, and had a small clear halo surrounding a partially stained basophilic internal structure. Hyphae were tapered on one end and had oval to pyriform swellings of 7-10 µm on the other, resulting in a bulbous appearance. Fungal elements stained positively with Gomori methenamine silver and Periodic acid-Schiff stains. The dog was euthanized at the owner's request, and necropsy revealed marked peripheral and visceral lymphadenomegaly. Histopathologic examination of lymph nodes confirmed granulomatous lymphadenitis with many fungal hyphae. Fungal culture yielded pure cultures of organisms that failed to produce ascospores or conidia precluding morphologic identification. PCR was performed using pan-fungal primers, ITS-1 and ITS-2, to amplify the intergenic spacer regions of ribosomal RNA; the PCR product was sequenced and a BLAST search of the GenBank databases at NCBI revealed 100% identity of the organism with Talaromyces helicus, the sexual form of Penicillium helicum. Talaromyces helicus has not previously been reported to cause disease in people or animals.

Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii.

Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 degrees C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The full-length m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.