Pulmonary dimorphic fungal infections among HIV/AIDS non-TB patients with chronic cough in Kampala, Uganda.

INTRODUCTION: Dimorphic fungi cause infection following the inhalation of spores into the pulmonary system. In the lower respiratory tract, the conidia transform into yeasts, which are engulfed by alveolar macrophages and may be destroyed without disease manifestation. However, in some immunocompromised individuals, they may persist and cause active fungal disease characterized by formation of granulomas in the infected tissues, which may mimic Mycobacterium tuberculosis (MTB). OBJECTIVE: To determine the prevalence of pulmonary dimorphic fungal infections among HIV/AIDS patients with non-TB chronic cough at Mulago National Referral and Teaching Hospital in Kampala, Uganda. METHODS: Sputum samples were collected from 175 consented HIV/AIDS patients attending the immuno-suppression syndrome (ISS) clinic at the hospital. Upon Xpert MTB/RIF sputum testing, 21 patients tested positive for MTB, and these were excluded from further analysis. The other 154 sputum negative samples were then subjected to PCR for dimorphic fungi at MBN Clinical Laboratories. Singleplex PCR was used to detect the target sequences in selected respective genes of each dimorphic fungal species of interest. DNA amplicons were detected based on gel electrophoresis. RESULTS: Dimorphic fungi were detected in 16.2% (25/154) of the studied population. Of these 9.1% (14/154) had Blastomyces dermatitidis and 7.1% (11/154) had Talaromyces marneffei. The remaining 84% of the studied participants had no dimorphic fungi. Histoplasma capsulatum, Coccidioides immitis and Paracoccidioides brasiliensis were not detected in any of the participants. CONCLUSION: Dimorphic fungi (B. dermatitidis and T. marneffei) were found in 16.2% of the HIV/AIDS patients with non-TB chronic cough in Kampala, Uganda. We recommend routine testing for these pathogens among HIV/AIDS patients with chronic cough.

Talaromides A-C, Bioactive Cyclic Heptapeptides from Talaromyces siglerae Isolated from a Marine Sponge.

Three new cyclic heptapeptides, talaromides A-C (1-3), were isolated from cultures produced by the fungus Talaromyces siglerae (Ascomycota), isolated from an unidentified sponge. The structures, featuring an unusual proline-anthranilic moiety, were elucidated by analysis of spectroscopic data and chemical transformations, including the advanced Marfey's method and GITC derivatization. Talaromides A and B inhibited migration activity against PANC-1 human pancreatic cancer cells without significant cytotoxicity.

Cytotoxic and Antibacterial Meroterpenoids Isolated from the Marine-Derived Fungus Talaromyces sp. M27416.

Three novel meroterpenoids, taladrimanins B-D (1-3), were isolated from the marine-derived fungus Talaromyces sp. M27416, alongside three biogenetically related compounds (4-6). We delineated taladrimanin B's (1) structure using HRESIMS and NMR, confirmed its configuration via quantum chemical NMR analysis and DP4+ methodology, and verified it through X-ray crystallography. ECD calculations determined the absolute configuration of compound 1, while comparative NMR and ECD analyses elucidated the absolute configurations of 2 and 3. These compounds are drimane-type meroterpenoids with a C10 polyketide unit (8R-configuration). We proposed a biosynthetic pathway and noted that compound 1 showed cytotoxic activity against MKN-45 and 5637 cell lines and selective antibacterial effects against Staphylococcus aureus CICC 10384.

Pullenvalenes A-D: Nitric Oxide-Mediated Transcriptional Activation (NOMETA) Enables Discovery of Triterpene Aminoglycosides from Australian Termite Nest-Derived Fungi.

We report on the use of nitric oxide-mediated transcriptional activation (NOMETA) as an innovative means to detect and access new classes of microbial natural products encoded within silent biosynthetic gene clusters. A small library of termite nest- and mangrove-derived fungi and actinomyces was subjected to cultivation profiling using a miniaturized 24-well format approach (MATRIX) in the presence and absence of nitric oxide, with the resulting metabolomes subjected to comparative chemical analysis using UPLC-DAD and GNPS molecular networking. This strategy prompted study of Talaromyces sp. CMB-TN6F and Coccidiodes sp. CMB-TN39F, leading to discovery of the triterpene glycoside pullenvalenes A-D (1-4), featuring an unprecedented triterpene carbon skeleton and rare 6-O-methyl-N-acetyl-d-glucosaminyl glycoside residues. Structure elucidation of 1-4 was achieved by a combination of detailed spectroscopic analysis, chemical degradation, derivatization and synthesis, and biosynthetic considerations.

Morphology, Development, and Pigment Production of Talaromyces marneffei are Diversely Modulated Under Physiologically Relevant Growth Conditions.

Talaromyces marneffei is an opportunistic pathogenic fungus that mainly affects HIV-positive individuals endemic to Southeast Asia and China. Increasing efforts have been made in the pathogenic mechanism and host interactions understanding of this pathogen in the last two decades; however, there are still no conclusions on how T. marneffei was transmitted from the donor bamboo rats to humans. A perception that the failure of fungus isolation from soil was attributed to the low salt tolerance of T. marneffei. Therefore, the effect of environmental fluctuations in fungal growth and development is fundamental for the characterization of its origin and fungal biology understanding. Herein, we characterized high osmolarity, pH, metal ions, nutrients, and oxidative stress have versatile effects on T. marneffei hyphal or yeast growth, conidia generation, and pigment production. Among these, high pH, low glucose amounts, and the inorganic nitrogen ammonium tartrate stimulated the red pigment production, whereas high osmolarity, high pH, and the inorganic nitrogen sodium nitrate could significantly accelerate the conidia generation. Specifically, zinc starvation repressed conidia generation and prevented the wrinkled yeast colony formation, indicating the function of zinc regulators in pathogenicity regulation. Since conidia are recognized as the infectious propagules, the effects characterization of different environmental factors in T. marneffei morphology in this work will not only expand the growth and pathogenic biology understanding of the fungus but also provide more clues for the T. marneffei infection transmission origin investigation.

New diarylcyclopentenone enantiomers and biphenyl derivatives from the fungus Talaromyces adpressus.

Ten new compounds, including three pairs of diarylcyclopentenone enantiomers (±) talaromycesins A-C (1-3) and four biphenyl derivatives talaromycesins D-G (4-7), along with four known compounds (8-11), were isolated from the fungus Talaromyces adpressus. Their structures were determined by analyses of extensive NMR spectroscopic and HRESIMS data, and their absolute configurations were elucidated by the dimolybdenum tetraacetate [Mo2(AcO)4]-induced ECD spectra, X-ray crystallographic studies, and ECD calculations. These new compounds were evaluated for their immunosuppressive activities for the first time, and compound 7 probably exerted liver-protective and anti-inflammatory effects on Con A-induced AIH by decreasing the levels of inflammatory cytokines, modulating immune homeostasis, and decreasing hepatocyte apoptosis, which may become a potential drug for the treatment of autoimmune diseases.

4-Hydroxy-2-pyridone derivatives with antitumor activity produced by mangrove endophytic fungus Talaromyces sp. CY-3.

OSMAC strategy is a useful tool for discovering series of metabolites from microorganism. Five new sambutoxin derivatives (1-2, 4, 8-9), together with seven known compounds (3, 5-7, 10-12), were isolated from Talaromyces sp. CY-3 under OSMAC strategy and guidance of molecular networking. Their planar structures and absolute configurations were determined by NMR, HRESIMS, ECD spectra and common biosynthetic pathway. In bioassay, compounds 1-12 showed cytotoxicity to tumor cell lines with IC50 values in the range of 1.76-49.13 µM. The antitumor molecular mechanism of 10 was also explored. In vitro compound 10 significantly inhibited the growth and proliferation of two lung cancer cell lines (A549 and H1703). Furthermore, colony formation, EdU analysis, flow cytometry and Western blot analysis showed that 10 could induce cell cycle arrest in G0/G1 phase by promoting the expression of p53 and p21. The molecular mechanism of its antitumor effects in vitro is that 10 arrests the cell cycle by activating the p21/CyclinD1/Rb signaling pathway and the p53 pathway. Our results identified a lead small molecule compound with efficient antitumor growth and proliferation activity.

Molecular and biological characterization of a novel partitivirus from Talaromyces pinophilus.

Talaromyces spp. have a worldwide distribution, are ecologically diverse and have been isolated from numerous different substrates. Talaromyces spp. are considered biotechnologically important due to their ability to produce a range of enzymes and pigments. Talaromyces pinophilus, belonging to genus Talaromyces and family Trichocomaceae, is known for producing several important bioactive metabolites. Here we report the isolation and characterisation of a partitivirus from T. pinophilus which we have nominated Talaromyces pinophilus partitivirus-1 (TpPV-1). TpPV-1 possesses a genome consisting of three double stranded (ds) RNA segments i.e., dsRNAs1-3, 1824 bp, 1638 bp and 1451 bp respectively, which are encapsidated in icosahedral particles 35 nm in diameter. Both dsRNA1 and dsRNA2 contain a single open reading frame (ORF) encoding respectively a 572 amino acid (aa) protein of 65 kDa and a 504 aa protein of 50 kDa. The third segment (dsRNA3) is potentially a satellite RNA. Phylogenetic analysis revealed that the TpPV-1 belongs to the family Partitiviridae in the proposed genus Zetapartitivirus. TpPV-1 infection decreases the mycelial growth rate of the host fungus and alters pigmentation as indicated by time course experiments performed on a range of different solid media comparing virus-infected and virus-free isogenic lines. This is the first report of mycovirus infection in T. pinophilus and may provide insights into understanding the effect of the mycovirus on the production of enzymes and pigments by the host fungus.

Isolation and characterization of a newly discovered plant growth-promoting endophytic fungal strain from the genus Talaromyces.

In the Kandi zone of Punjab, India, root and rhizospheric soil samples were collected from the local vegetation near the Shivalik mountain foothills. Fifteen fungal colonies exhibiting distinct cultural morphology on Potato Dextrose Agar (PDA) plates were selected for plant-microbe interaction studies. Among these, the isolate HNB9 was identified as a nonpathogenic root colonizer. Morphological and molecular analyses confirmed HNB9 as Talaromyces albobiverticillius, characterized by the secretion of a red pigment as a secondary metabolite. Plants colonized with T. albobiverticillius HNB9 exhibited enhanced growth, manifesting in increased shoot and root length compared to untreated controls. This study unveiled the first evidence that a species from the Talaromyces genus, specifically T. albobiverticillius, possesses dual capabilities of root colonization and plant growth promotion. Moreover, HNB9 demonstrated the production of plant growth-regulating compounds like Indole Acetic Acid (IAA) and proficient solubilization of crucial nutrients (Phosphorous, Zinc, and Silica) through plate culture methods. This finding represents a significant contribution to the understanding of root-colonizing fungi with plant growth-promoting attributes, challenging the existing knowledge gap within the Talaromyces genus.

Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

Talaromyces marneffei endocarditis initially detected by Next Generation Sequencing: A case report.

BACKGROUND: Talaromyces marneffei (T. marneffei) is a thermal dimorphic fungus, which can cause lung or blood stream infection in patients, often life-threatening. However, endocarditis caused by T. marneffei has not been reported. For elderly patients with implanted cardiac devices or artificial valves, the prevention and treatment of infective endocarditis should not be ignored. METHODS: This is a descriptive study of a T. marneffei endocarditis by joint detection of cardiac ultrasound examination, peripheral blood DNA metagenomics Next Generation Sequencing (mNGS), and in vitro culture. RESULTS: We describe an 80-year-old female patient with an unusual infection of T. marneffei endocarditis. After intravenous drip of 0.2 g voriconazole twice a day for antifungal treatment, the patient showed no signs of improvement and their family refused further treatment. CONCLUSION: Infective endocarditis is becoming more and more common in the elderly due to the widely use of invasive surgical procedures and implantation of intracardiac devices. The diagnosis and treatment of T. marneffei endocarditis is challenging because of its rarity. Here, we discussed a case of T. marneffei endocarditis, and emphasized the role of mNGS in early diagnosis, which is of great significance for treatment and survival rate of patients.

In vivo assembly of genetic constructs in filamentous fungus Talaromyces cellulolyticus.

In the filamentous fungus Talaromyces cellulolyticus, similar to other filamentous fungi, non-homologous recombination predominates over homologous recombination. For instance, to achieve an acceptable integration frequency of a genetic construct into a target site on the intact chromosome, the flanking sequences directing this integration should be approximately 2.5 kb in length. However, despite the requirement of long flanks for integration into the intact chromosome, we found that homologous recombination between linear DNA fragments in T. cellulolyticus effectively occurs when these fragments overlap by just 50 bp. This allows for the assembly of full-sized genetic constructs in vivo from relatively small blocks, eliminating the need for in vitro assembly, similar to the approach previously developed for the yeast Saccharomyces cerevisiae. To validate this possibility, we replaced the native promoter of the target gene by transforming the recipient strain with five DNA fragments: two flanks for recombination with the target locus, two parts of the marker gene, and a donor promoter. This discovery significantly expedites the genetic engineering of T. cellulolyticus and potentially other fungi.

Talaromyces sedimenticola sp. nov., isolated from the Mariana Trench.

Two fungal strains (K-2T and S1) were isolated from the deepest ocean sediment of the Challenger Deep located in the Mariana Trench. The internal transcribed spacer (ITS) gene sequences of the isolates K-2T and S1 differed from those of closely related species, such as Talaromyces assiutensis and T. trachyspermus. Phylogenetic analyses based on single and concatenated alignments of the genes, namely ITS, ß-tubulin (benA), calmodulin (cam), and the second-largest subunit fragment of the RNA polymerase II (rpb2) showed that the isolates K-2T and S1 were clustered together with other Talaromyces species, such as T. trachyspermus and T. assiutensis, as evidenced by the position on a terminal branch with high bootstrap support. They could also be distinguished from their closest relatives with valid published names via morphological and physiological characteristics, for example, growth at 4 °C-50 °C with a pH in the range of 1.5-12. Based on their phylogenetic, morphological, and physicochemical properties, the isolates K-2T and S1 represent a novel species in the genus Talaromyces, and the proposed name is Talaromyces sedimenticola sp. nov. The type strain is K-2T (= GDMCC 3.746T = JCM 39451T).

Talaromyces mellisjaponici sp. nov., a xerophilic species isolated from honey in Japan.

Five isolates of a xerophilic Talaromyces species were obtained from honey in Japan. Molecular phylogenetic analysis based on a combined dataset for four regions (rRNA internal transcribed spacer, ß-tubulin, calmodulin and RNA polymerase II second largest subunit) revealed that the strains formed an independent clade in section Trachyspermi, which is sister to Talaromyces affinitatimellis, Talaromyces basipetosporus and Talaromyces speluncarum. The strains and their relatives have different growth on creatine agar, yeast extract sucrose agar and dichloran 18 % glycerol agar, different branching patterns (mostly monoverticillate or biverticillate, less frequently divaricate or terverticillate), and different sizes and surface structures of conidia. Xerotolerance tests were also conducted using media adjusted to five different sucrose concentrations (0, 20, 40, 60 and 80 %). The colony diameters of the strains were larger than those of T. affinitatimellis, T. basipetosporus and T. speluncarum at each sucrose concentration. Altogether, the obtained morphological, molecular and physiological data allowed the proposal of Talaromyces mellisjaponici sp. nov. for this novel species, with NBRC 116048T as the type strain.

New Polyene Macrolide Compounds from Mangrove-Derived Strain Streptomyces hiroshimensis GXIMD 06359: Isolation, Antifungal Activity, and Mechanism against Talaromyces marneffei.

Mangrove-derived actinomycetes represent a rich source of novel bioactive natural products in drug discovery. In this study, four new polyene macrolide antibiotics antifungalmycin B-E (1-4), along with seven known analogs (5-11), were isolated from the fermentation broth of the mangrove strain Streptomyces hiroshimensis GXIMD 06359. All compounds from this strain were purified using semi-preparative HPLC and Sephadex LH-20 gel filtration while following an antifungal activity-guided fractionation. Their structures were elucidated through spectroscopic techniques including UV, HR-ESI-MS, and NMR. These compounds exhibited broad-spectrum antifungal activity against Talaromyces marneffei with minimum inhibitory concentration (MIC) values being in the range of 2-128 µg/mL except compound 2. This is the first report of polyene derivatives produced by S. hiroshimensis as bioactive compounds against T. marneffei. In vitro studies showed that compound 1 exerted a significantly stronger antifungal activity against T. marneffei than other new compounds, and the antifungal mechanism of compound 1 may be related to the disrupted cell membrane, which causes mitochondrial dysfunction, resulting in leakage of intracellular biological components, and subsequently, cell death. Taken together, this study provides a basis for compound 1 preventing and controlling talaromycosis.

Enantiomeric α-pyrone derivatives with immunosuppressive activity from Talaromyces adpressus.

Five pairs of undescribed enantiomeric α-pyrone derivatives (±)-adprepyrones A-E (±1-±5), together with an unreported congener adprepyrone F (6), and 6-[(E)-3-Hydroxyprop-1-enyl]-4-methoxy-5-methyl-2-pyrone (7), recently reported as synthetic compound, were isolated from the fungus Talaromyces adpressus. Their structures with absolute configurations were elucidated by HRESIMS, 1D and 2D NMR, electronic circular dichroism calculations, and single-crystal X-ray diffraction analyses. (±)-Adprepyrone A (±1) possesses an unreported carbon skeleton formed by the fusion of an α-pyrone derivative with nicotinamide. Compounds (+)-2, (±)-4, (±)-5, and 7 showed moderate inhibitory activity against concanavalin A (ConA)-induced T lymphocyte proliferation with IC50 values ranging from 8.9 to 19.8 µM.

Talcarpones A and B: bisnaphthazarin-derived metabolites from the Australian fungus Talaromyces johnpittii sp. nov. MST-FP2594.

Talcarpones A (1) and B (2) are rare bisnaphthazarin derivatives produced by Talaromyces johnpittii (ex-type strain MST-FP2594), a newly discovered Australian fungus, which is formally described and named herein. The talcarpones were isolated along with the previously reported monomeric naphthoquinone, aureoquinone (3), suggesting a biosynthetic link between these metabolites. Talcarpone A is a lower homologue of hybocarpone (4), which was first isolated from a mycobiont of the lichen Lecanora hybocarpa. The structures of 1 and 2 were elucidated by detailed spectroscopic analysis, molecular modelling and comparison with literature data. Talcarpones 1 and 2 exhibited moderate antifungal activity (MIC 0.78-3.1 µg ml-1) and weak activity against Gram-positive bacteria (MIC 13-25 µg ml-1). The talcarpones also demonstrated noteworthy chemical reactivities, with 2 converting rapidly to 1, which in turn converted slowly to the highly coloured 3. These post-biosynthetic reactions point to a potential ecological role for the talcarpones in providing ongoing (slow-release) physicochemical protection for T. johnpittii against solar irradiation.

New Dipyrroloquinones from a Plant-Derived Endophytic Fungus Talaromyces sp.

Two new dipyrroloquinones, namely talaroterreusinones A (1) and B (2), together with four known secondary metabolites, terreusinone A (3), penicillixanthone A (4), isorhodoptilometrin (5), and chrysomutanin (6), were isolated from the solid culture of the endophytic fungus Talaromyces sp. by integrating mass spectrometry-based metabolic profiling and a bioassay-guided method. Their planar structures and stereochemistry were elucidated by comprehensive spectroscopic analysis including NMR and MS. The absolute configuration at C-1″ of terreusinone A (1) was established by applying the modified Mosher's method. Compounds 1-6 were evaluated for anti-inflammatory activity and cytotoxicity. As a result, 1-3 inhibited the LPS-stimulated NO production in macrophage RAW264.7 cells, with IC50 values of 20.3, 30.7, and 20.6 µM, respectively. Penicillixanthone A (4) exhibited potent cytotoxic activity against Hep G2 and A549 cell lines, with IC50 values of 117 nM and 212 nM, respectively, and displayed significant antitumour effects in A549 cells by inhibiting the PI3K-Akt-mTOR signalling pathway.

[From treatment to whole course management: envisioning comprehensive management of Talaromycosis marneffei].

Talaromycosis marneffei has been increasing in recent years. Our understanding of this disease has gradually deepened through extensive basic and clinical research, but there are still many limitations. In this article, by incorporating the latest research advancements, we discuss important issues in managing Talaromycosis marneffei trends, aiming to guide effective prevention and control of the disease, improving public health, and reducing the healthcare burden.

Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression.

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A-E (3-7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2-C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.