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Talaromyces marneffei is a thermally dimorphic fungus that can cause life-threatening systemic mycoses, particularly in immunocompromised individuals. Fungal homeobox transcription factors control various developmental processes, including the regulation of sexual reproduction, morphology, metabolism, and virulence. However, the function of homeobox proteins in T. marneffei has not been fully explored. Here, we searched the T. marneffei genome for the total homeobox transcription factors and predicted their biological relevance by performing gene expression analysis in different cell types, including conidia, mycelia, yeasts, and during phase transition. RfeB is selected for further computational analysis since (i) its transcripts were differentially expressed in different phases of T. marneffei , and (ii) this protein contains the highly conserved protein-protein interaction region (IR), which could be important for pathobiology and have therapeutic application. To assess the structure-function of the IR region, in silico alanine substitutions were performed at three-conserved IR residues (Asp276, Glu279, and Gln282) of RfeB, generating a triple RfeB mutated protein. Using 3D modeling and molecular dynamics simulations, we compared the protein complex formation of wild-type and mutated RfeB proteins with the putative partner candidate TmSwi5. Our results demonstrated that the mutated RfeB protein exhibited increased free binding energy, elevated protein compactness, and a reduced number of atomic contacts, suggesting disrupted protein stability and interaction. Notably, our model revealed that the IR residues primarily stabilized the RfeB binding sites located in the central region (CR). This computational approach for protein mutagenesis could provide a foundation for future experimental studies on the functional characterization of RfeB and other homeodomain-containing proteins in T. marneffei .

Talaromycosis is a fungal infection caused by an opportunistic dimorphic fungus Talaromyces marneffei. During infection, T. marneffei resides inside phagosomes of human host macrophages where the fungus encounters nutrient scarcities and host-derived oxidative stressors. Previously, we showed that the deletion of acuK, a gene encoding Zn(2)Cys(6) transcription factor, caused a decreased ability for T. marneffei to defend against macrophages, as well as a growth impairment in T. marneffei on both low iron-containing medium and gluconeogenic substrate-containing medium. In this study, a paralogous gene acuM was deleted and characterized. The ΔacuM mutant showed similar defects with the ΔacuK mutant, suggesting their common role in gluconeogenesis and iron homeostasis. Unlike the pathogenic mold Aspergillus fumigatus, the ΔacuK and ΔacuM mutants unexpectedly exhibited normal siderophore production and did not show lower expression levels of genes involved in iron uptake and siderophore synthesis. To identify additional target genes of AcuK and AcuM, RNA-sequencing analysis was performed in the ΔacuK and ΔacuM strains growing in a synthetic dextrose medium with 1% glucose at 25 °C for 36 hours. Downregulated genes in both mutants participated in iron-consuming processes, especially in mitochondrial metabolism and anti-oxidative stress. Importantly, the ΔacuM mutant was sensitive to the oxidative stressors menadione and hydrogen peroxide while the ΔacuK mutant was sensitive to only hydrogen peroxide. The yeast form of both mutants demonstrated a more severe defect in antioxidant properties than the mold form. Moreover, ribosomal and ribosomal biogenesis genes were expressed at significantly lower levels in both mutants, suggesting that AcuK and AcuM could affect the protein translation process in T. marneffei. Our study highlighted the role of AcuK and AcuM as global regulators that control multiple cellular adaptations under various harsh environmental conditions during host infection. These transcription factors could be potentially exploited as therapeutic targets for the treatment of this neglected infectious disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wangsanut et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

β-Thalassemia is an inherited genetic disorder associated with β-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of β-thalassemia. This study conducted a virtual screening of certain compounds similar to 5'-deoxy-5'methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of β-thalassemia.

Antibody immunity is now known to play a critical role in combating mycotic infections. The identification of molecules that can elicit an antibody response against fungal pathogens is the first step in developing antibody-based therapeutic strategies. Antigenic proteins are molecules recognized by the immune system that can stimulate antibody production and, therefore, can be a direct target for studying human-fungal pathogen interactions. Advances in recent immunoproteomic approaches have substantially aided in determining the key antigenic proteins on a large scale. In this review, we present a collection of antigenic proteins identified in yeast, dimorphic, and filamentous fungal pathogens to date. The general features of antigenic proteins are summarized and reveal that the proteins could commonly function in antistress responses, protein synthesis, and metabolism. The antigenic proteins listed here could serve as starting materials for developing species-specific or broad-spectrum diagnostic tests, therapeutic antibodies, and even vaccines against fungal infections., Competing Interests: The authors declare no conflict of interest.

Background and Aim: Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli , Pseudomonas aeruginosa , Bacillus cereus , and Staphylococcus aureus , which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals., Materials and Methods: After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 μg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli , P. aeruginosa , B. cereus , and S. aureus , using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum . Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus ., Results: Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 μg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa , B. cereus , and E. coli , at 256 and >256 μg/mL, respectively. We observed that at least 256 μg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16-128 μg/mL, it exhibited antibiofilm activity against S. aureus . Furthermore, at 256 μg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus . Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 μg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 μg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 μg/mL., Conclusion: Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Rodjan, et al.)

Talaromyces marneffei is a human fungal pathogen that causes endemic opportunistic infections, especially in Southeast Asia. The key virulence factors of T. marneffei are the ability to survive host-derived heat and oxidative stress, and the ability to convert morphology from environmental mold to fission yeast forms during infection. Glutathione metabolism plays an essential role in stress response and cellular development in multiple organisms. However, the role of the glutathione system in T. marneffei is elusive. Here, we identified the genes encoding principal enzymes associated with glutathione metabolism in T. marneffei, including glutathione biosynthetic enzymes (Gcs1 and Gcs2), glutathione peroxidase (Gpx1), glutathione reductase (Glr1), and a family of glutathione S-transferase (Gst). Sequence homology search revealed an extended family of the TmGst proteins, consisting of 20 TmGsts that could be divided into several classes. Expression analysis revealed that cells in conidia, mold, and yeast phases exhibited distinct expression profiles of glutathione-related genes. Also, TmGst genes were highly upregulated in response to hydrogen peroxide and xenobiotic exposure. Altogether, our findings suggest that T. marneffei transcriptionally regulates the glutathione genes under stress conditions in a cell-type-specific manner. This study could aid in understanding the role of glutathione in thermal-induced dimorphism and stress response., (© 2023. Springer Nature Limited.)

Talaromycosis is a fungal infection that generally affects immunocompromised hosts and is one of the most frequent systemic mycoses in HIV patients, especially in endemic areas such as Southeast Asia. Talaromyces marneffei , the causative agent of talaromycosis, grows as a mold in the environment but adapts to the human body and host niches by transitioning from conidia to yeast-like cells. Knowledge of the human host and T. marneffei interaction has a direct impact on the diagnosis, yet studies are still lacking. The morbidity and mortality rates are high in taloromycosis patients if the diagnosis and treatments are delayed. Immunogenic proteins are excellent candidates for developing detection tools. Previously, we identified antigenic proteins that were recognized by antibodies from talaromycosis sera. Three of these identified proteins have been previously characterized in detail, while the others have not been explored. To expedite the progress of antigen discovery, the complete list of antigenic proteins and their features was fully reported in this study. Functional annotation and Gene Ontology examination revealed that these proteins showed a high association with membrane trafficking. Further bioinformatics analyses were performed to search for antigenic protein characteristics, including functional domains, critical residues, subcellular localization, secretory signals, and epitope peptide sequences. Expression profiling of these antigenic encoding genes was investigated using quantitative real-time PCR. The results demonstrated that most genes were expressed at low levels in the mold form, but were highly upregulated in the pathogenic yeast phase, consistent with the antigenic role of these genes during the human-host interaction. Most transcripts accumulated in the conidia, suggesting a role during phase transition. The collection of all antigen-encoding DNA sequences described here is freely accessible at GenBank, which could be useful for the research community to develop into biomarkers, diagnostic tests, research detection tools, and even vaccines., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wangsanut, Amsri and Pongpom.)

Background and Aim: Fungal zoonoses are an economic and public health concern because they can cause various degrees of morbidity and mortality in animals and humans. To combat this issue, alternative natural antifungals, such as products derived from rice protein hydrolysates or rice antifungal protein/peptide are being considered because they are highly bioactive and exhibit various functional properties. Thailand is a leading rice producer and exporter. Among the various cultivated rice varieties, Sangyod rice ( Oryza sativa L.) is exclusively indigenous to Thailand's Phatthalung province; it has a Thai geographical indication tag. Here, we investigated whether the Phatthalung Sangyod rice seeds have bioactive antifungal peptides., Materials and Methods: Antifungal activity in four Sangyod rice seed extracts (SYPs) - namely, (1) the crude lysate, SYP1; (2) the heat-treated lysate, SYP2; (3) the heat- and pepsin digested lysate, SYP3; and (4) the heat- and proteinase K-digested lysate, SYP4 - was analyzed. Protein concentrations in these SYPs were determined using the Bradford assay. The total phenolic compound content was determined using the modified Folin-Ciocalteu method in a 96-well microplate. Then, the SYP protein pattern was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, using the agar well diffusion method, the antifungal properties of these SYPs were tested against ten medically important pathogenic fungi. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration values were determined for the active SYPs - SYP2-4. Finally, the clinical safety of SYP4 was determined using a hemolytic assay (using canine red blood cells [RBCs])., Results: The crude lysate SYP1 did not show antifungal activity against any of the ten tested pathogenic fungi. Surprisingly, hydrolysates SYP2, SYP3, and SYP4 displayed antifungal properties against the ten tested pathogenic fungi. Thus, heat and enzymatic hydrolysis seem to transform the bioactivity of the crude protein extract - SYP1. Further, SYP4 shows the most effective antifungal activity. It completely inhibited Cryptococcus neoforman s, Talaromyces marneffei yeast phase, Trichophyton mentagrophyte s, and Trichophyton rubru m. A partial inhibitory action on Candida albican s and Microsporum gypseum was possessed while showing the least activity to C. neoforman s. SYP4 was nontoxic to canine RBCs. Hemolysis of canine RBCs was undetectable at 1 × MIC and 2 × MIC concentrations; therefore, it can be safely used in further applications., Conclusion: These results indicate that heat and proteinase K hydrolyzed SYP is a very potent antifungal preparation against animal and human fungal pathogens and it can be used in future pharmaceuticals and functional foods., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Jeenkeawpieam, et al.)

Siderophores are compounds with low molecular weight with a high affinity and specificity for ferric iron, which is produced by bacteria and fungi. Fungal siderophores have been characterized and their feasibility for clinical applications has been investigated. Fungi may be limited in slow growth and low siderophore production; however, they have advantages of high diversity and affinity. Hence, the purpose of this study was to generate a genetically modified strain in Talaromyces marneffei that enhanced siderophore production and to identify the characteristics of siderophore to guide its medical application. SreA is a transcription factor that negatively controls iron acquisition mechanisms. Therefore, we deleted the sreA gene to enhance the siderophore production and found that the null mutant of sreA (Δ sreA ) produced a high amount of extracellular siderophores. The produced siderophore was characterized using HPLC-MS, HPLC-DAD, FTIR, and 1 H- and 13 C-NMR techniques and identified as a coprogen B. The compound showed a powerful iron-binding activity and could reduce labile iron pool levels in iron-loaded hepatocellular carcinoma (Huh7) cells. In addition, the coprogen B showed no toxicity to the Huh7 cells, demonstrating its potential to serve as an ideal iron chelator. Moreover, it inhibits the growth of Candida albicans and Escherichia coli in a dose-dependent manner. Thus, we have generated the siderophore-enhancing strain of T . marneffei , and the coprogen B isolated from this strain could be useful in the development of a new iron-chelating agent or other medical applications.

Talaromyces ( Penicillium ) marneffei is an important dimorphic mycosis endemic in Southeast Asia and Southern China, but the origin and maintenance of virulence traits in this organism remains obscure. Several pathogenic fungi, including Cryptococcus neoformans , Aspergillus fumigatus, Blastomyces dermatitidis , Sporothrix schenckii , Histoplasma capsulatum and Paracoccidioides spp. interact with free living soil amoebae and data suggests that fungal pathogenic strategies may emerge from environmental interactions of these fungi with ubiquitous phagocytic microorganisms. In this study, we examined the interactions of T. marneffei with the soil amoeba Acanthamoeba castellanii . T. marneffei was rapidly ingested by A. castellanii and phagocytosis of fungal cells resulted in amoeba death after 24 h of contact. Co-culture also resulted in a rapid transition for conidia to the fission-yeast form. In addition, well-established virulence factors such as melanin and a yeast specific mannoprotein of T. marneffei were expressed during interaction with A. castellanii at 37°C. Our findings support the assumption that soil amoebae environmental predators play a role in the selection and maintenance of particular features in T. marneffei that impart virulence to this clinically important dimorphic fungus in mammalian hosts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pruksaphon, Nosanchuk, Thammasit, Pongpom and Youngchim.)

Morphogenesis and stress adaptation are key attributes that allow fungal pathogens to thrive and infect human hosts. During infection, many fungal pathogens undergo morphological changes, and this ability is highly linked to virulence. Furthermore, pathogenic fungi have developed multiple antioxidant defenses to cope with the host-derived oxidative stress produced by phagocytes. Glutathione is a major antioxidant that can prevent cellular damage caused by various oxidative stressors. While the role of glutathione in stress detoxification is known, studies of the glutathione system in fungal morphological switching and virulence are lacking. This review explores the role of glutathione metabolism in fungal adaptation to stress, morphogenesis, and virulence. Our comprehensive analysis of the fungal glutathione metabolism reveals that the role of glutathione extends beyond stressful conditions. Collectively, glutathione and glutathione-related proteins are necessary for vitality, cellular development and pathogenesis.

Talaromyces marneffei is a dimorphic pathogenic fungus causing opportunistic infection in immunocompromised patients. It is a facultative intracellular pathogen and is usually found inside the host macrophages during infection. Alternative carbons and iron are the important nutrients associated with intracellular survival and pathogenesis of T. marneffei . This study reported the importance of the transcription factor AcuK in control of gluconeogenesis and iron acquisition in T. marneffei . Deletion of acuK gene in T. marneffei resulted in retardation of growth and germination in both mold and yeast phases. Microscopically, Δ acuK showed double nuclei hyphae. However, the yeast cells showed normal morphology. The Δ acuK failed to grow in iron-limiting conditions. Additionally, it could not grow in a medium containing gluconeogenic carbon sources. Moreover, Δ acuK showed higher susceptibility to macrophage killing than the wild type. These results demonstrated that AcuK controlled both iron acquisition and gluconeogenesis, and it could contribute to the pathogenicity of this fungus.

Purpose: To evaluate etiology and prognostic factors for treatment outcomes of fungal keratitis (FK)., Methods: Culture-positive FK patients between 2012 and 2017 were reviewed. Treatment outcomes were categorized into success (resolved within two weeks), slow response and medication failure (no improvement or required surgery). Etiology and risk factors for poor treatment outcomes were analyzed., Results: A total of 113 eyes of 113 patients (77% males) were recruited. Ocular trauma (69.0%) was the most common predisposing factor. Of this, 80% were exposed to organic foreign bodies. The most common pathogen was Fusarium spp. (45.2%), while dematiaceous fungi were discovered in 29.6%. Medical treatment was successful in 24.8% of eyes, while 29.2% had a slow response and 42.5% failed medication. Therapeutic keratoplasty was performed in 22.1% of eyes and 11.5% eventually required eye removal. Significant risk factors for medication failure were advanced age (P = 0.005), delayed antifungal treatment (P = 0.038) and large-size lesion (P = 0.003)., Conclusions: Ocular trauma was the major predisposing factor of FK in the Northern Thailand. Fusarium was the most common identified pathogen. Many cases were refractory to medications and required surgical intervention. Aging, delayed treatment and a large lesion were predictors for poor outcomes., Competing Interests: Declaration of Competing Interest No conflicting relationship exists for any author., (Copyright © 2021. Published by Elsevier Ltd.)

Dematiaceous fungal keratitis is an important etiology of visual loss, particularly in an agricultural society. From a retrospective review of medical records from 2012 to 2020, 50 keratitis cases of cultured-positive for dematiaceous fungi were presented at a tertiary care hospital in Northern Thailand. The study aimed to identify the isolated causative dematiaceous species using the PCR technique and to explore their related clinical features, including treatment prognoses. Sequencing of the amplified D1/D2 domains and/or ITS region were applied and sequenced. Of the 50 dematiaceous fungal keratitis cases, 41 patients were males (82%). In most cases, the onset happened during the monsoon season (June to September) (48%). The majority of the patients (72%) had a history of ocular trauma from an organic foreign body. The most common species identified were Lasiodiplodia spp. (19.35%), followed by Cladosporium spp. and Curvularia spp. (12.90% each). About half of the patients (52%) were in the medical failure group where surgical intervention was required. In summary, ocular trauma from an organic foreign body was the major risk factor of dematiaceous fungal keratitis in Northern Thailand. The brown pigmentation could be observed in only 26%. Significant prognostic factors for medical failure were visual acuity at presentation, area of infiltrate, depth of the lesions, and hypopyon.

Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, Rhinacanthus nasutus, against the human pathogenic fungus Talaromyces marneffei . This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The R. nasutus crude protein extract inhibited the growth of T. marneffei . The anti- T. marneffei activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from R. nasutus was partially purified by size fractionation to ≤10, 10-30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti- T. marneffei activity with the MIC and MFC values of 32 to 128 μg/mL and >128 μg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with T. marneffei mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti- T. marneffei activity of the 10-30 kDa fraction was abrogated by deletion of gasA and gasC , the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from R. nasutus represent sources for novel drug development.

Talaromyces marneffei is an opportunistic, dimorphic fungal pathogen that causes a disseminated infection in people with a weakened immunological status. The ability of this fungus to acquire nutrients inside the harsh environment of the macrophage phagosome is presumed to contribute to its pathogenicity. The transcription factors AcuM and AcuK are known to regulate gluconeogenesis and iron acquisition in Aspergillus fumigatus . This study demonstrated that they are also involved in both of these processes in the dimorphic fungus T. marneffei . Expression of acuM and acuK genes was determined by real time-polymerase chain reaction (RT-PCR) on the cells grown in media containing gluconeogenic substrates and various iron concentrations. We found that the acuM and acuK transcript levels were sequentially reduced when growing the fungus in increasing amounts of iron. The acuM transcript was upregulated in the gluconeogenic condition, while the acuK transcript showed upregulation only in the acetate medium in the yeast phase. These results suggest the involvement of acuM and acuK in gluconeogenesis and iron homeostasis in T. marneffei .

The pathogenic dimorphic fungus Talaromyces marneffei is known to cause a fatal systemic mycosis in immunocompromised patients, especially in HIV patients in Southeast Asia. The basic leucine-zipper (bZip) transcription factor gene, yapA, has been identified in T. marneffei. A prior study described that yapA was involved in the oxidative and nitrosative stress response in T. marneffei. Interestingly, an essential role of Saccharomyces cerevisiae Yap1p in the oxidative stress response is the activation of the transcription of its target genes. To identify the target genes of yapA in T. marneffei, the qRT-PCR method were used in this study. Investigation into the expression of genes which are probably regulated by yapA revealed that yapA controlled the expression of cat1 (catalase), cpeA (catalase-peroxidase), sodA (copper, zinc superoxide dismutase), gcs1 (glutamate-cysteine ligase), glr1 (glutathione oxidoreductase), trr1/trr2 (thioredoxin reductase), and trxA (thioredoxin) during stress conditions in all forms of conidium, mycelium, and yeast phase. An exception to this was the expression of cat1 under conditions of oxidative stress in the mould phase with a similar relative expression level in all of the wild-type, mutant and complemented strains. These genes are involved in response against oxidative stress and nitrosative stress in this fungus. The data showed that they could be regulated by the yapA gene during stress conditions. Moreover, the yapA gene is also known to control red pigment production by inhibiting the regulation of the five polyketide synthase (pks) genes, pks3 (polyketide synthase), rp1 (transcription activator), rp2 (β-subunit fatty acid synthase), rp3 (α-subunit fatty acid synthase), and rp4 (oxidoreductase) in the mould phase. In addition, it also regulates transcription in the laccase gene cluster including lac (extracellular dihydrogeodin oxidase/laccase), and multicopper oxidase encoding genes (PMAA_050860, PMAA_072680, PMAA_085520, PMAA_082010, and PMAA_082060) in all stages of the T. marneffei lifecycle (conidia, mould, and yeast phase). This study suggests the importance of the role of the yapA gene in the stress response and virulence of T. 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., Competing Interests: Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

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., Competing Interests: The authors have declared that no competing interests exist.

Talaromyces marneffei (or Penicillium marneffei) is an opportunistic pathogen that can cause disseminated disease in human immunodeficiency virus (HIV)-infected patients, especially in Southeast Asia. T. marneffei is a thermally dimorphic fungus. Typically, T. marneffei has an adaptive morphology. It grows in a filamentous form (mould) at 25°C and can differentiate to produce asexual spores (conidia). In contrast, at 37°C, it grows as yeast cells that divide by fission. This study aimed to validate a quantitative reverse-transcription polymerase chain reaction (qRT-PCR) for gene expression analysis in T. marneffei. Analysis of relative gene expression by qRT-PCR requires normalization of data using a proper reference gene. However, suitable reference genes have not been identified in gene expression studies across different cell types or under different experimental conditions in T. marneffei. In this study, four housekeeping genes were selected for analysis: β-actin (act); glyceraldehyde-3-phosphate dehydrogenase (gapdh); β-tubulin (benA) and 18S rRNA. Two analysis programs; BestKeeper and geNorm software tools were used to validate the expression of the candidate normalized genes. The results indicated that the actin gene was the one which was most stably expressed and was recommended for use as the endogenous control for gene expression analysis of all growth forms in T. marneffei by qRT-PCR under normal and stress conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In Aspergillus nidulans, the AcuK and AcuM transcription factors form a complex that regulates gluconeogenesis. In Aspergillus fumigatus, AcuM governs gluconeogenesis and iron acquisition in vitro and virulence in immunosuppressed mice. However, the function of AcuK was previously unknown. Through in vitro studies, we found that A. fumigatus ΔacuK single and ΔacuK ΔacuM double mutants had impaired gluconeogenesis and iron acquisition, similar to the ΔacuM mutant. Also, the ΔacuK, ΔacuM, and ΔacuK ΔacuM mutants had similar virulence defects in mice. However, the ΔacuK mutant had a milder defect in extracellular siderophore activity and induction of epithelial cell damage in vitro than did the ΔacuM mutant. Moreover, overexpression of acuM in the ΔacuK mutant altered expression of 3 genes and partially restored growth under iron-limited conditions, suggesting that AcuM can govern some genes independently of AcuK. Although the ΔacuK and ΔacuM mutants had very similar transcriptional profiles in vitro, their transcriptional profiles during murine pulmonary infection differed both from their in vitro profiles and from each other. While AcuK and AcuM governed the expression of only a few iron-responsive genes in vivo, they influenced the expression of other virulence-related genes, such as hexA and dvrA. Therefore, in A. fumigatus, while AcuK and AcuM likely function as part of the same complex, they can also function independently of each other. Furthermore, AcuK and AcuM have different target genes in vivo than in vitro, suggesting that in vivo infection stimulates unique transcriptional regulatory pathways in A. fumigatus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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 Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Penicillium marneffei is a significant opportunistic fungal pathogen in Southeast Asia and its ability to survive inside the host macrophages is believed to be important in the establishment of infection. Previously, we isolated a gene encoding a catalase- peroxidase (cpeA) from P. marneffei and showed that the cpeA transcript is specifically upregulated during yeast phase growth at 37 °C. In this study, the cpeA transcript was found to be induced during the mycelium to yeast phase transition and during stress conditions induced by hydrogen peroxide treatment. Null mutation of cpeA reduced the fungal tolerance to hydrogen peroxide but not to heat stress. These results indicated that the CpeA plays a crucial role in this fungus' oxidative stress response. Western blot analysis demonstrated that the CpeA induced antibody production in P. marneffei-infected patients, including highly exposed-healthy people. This is the first report that the catalase-peroxidase possesses an immunogenic property in fungi.

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.

A gene encoding an antigenic mannoprotein of Penicillium marneffei, MPLP6, was isolated by an antibody screening approach and characterized. The polypeptide chain containing deduced 220 amino acids has a predicted molecular mass of 24 kDa. It has high similarity to Mp1p, the first mannoprotein antigen isolated from P. marneffei. The polypeptide sequence presents the property of cell wall mannoproteins by containing a putative N-terminal signal peptide and potential O-linked glycosylation sites. However, absence of a GPI-anchored signal sequence suggested that this protein is secreted. The MPLP6 transcript was present specifically in the pathogenic yeast form. The transcript was completely absent in the mold phase and conidia. The fusion protein produced in E. coli was Western immunoblotted with P. marneffei-infected human sera and 95% of the patients' sera were positive in the assay. None of the sera obtained from patients with aspergillosis, tuberculosis, histoplasmosis or cryptococcosis tested positive. These results suggest that Mplp6 can be used as a marker in a serodiagnostic assay.

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.

We previously reported a nested PCR assay for specific identification of 18S ribosomal DNA of Penicillium marneffei. In this study, the assay was used to detect the DNA of P. marneffei in serum samples. Sensitivity of the test was 4 pg/microl and 0.4 fg/microl when the cycle numbers used for nested reactions were 15 and 30, respectively. Twenty four out of 35 sera (68.6%) collected from patients with culture confirmed penicilliosis marneffei were positive, while normal healthy and non-P. marneffei infected HIV-positive sera were negative. The results suggested that the assay could be applied for the diagnosis of infections due to P. marneffei.