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Background: This study evaluated the efficacy of fluconazole prophylaxis in reducing radiation-induced oral mucositis (RIOM) and Candida carriage in head and neck cancer (HNC) patients undergoing concurrent chemoradiotherapy (CCRT)., Methods: A prospective, double-blinded, randomized controlled trial was conducted with 78 HNC patients receiving either fluconazole (100 mg daily) or placebo during CCRT. The primary outcome was the incidence of grade 2 or higher RIOM. Secondary outcomes included positive Candida cultures and colony-forming units (CFUs). Mixed-effects ordinal logistic regression and logistic regression were used for analysis., Results: Fluconazole significantly reduced the incidence of grade 2 or higher RIOM at week 7 (p = 0.039), positive Candida carriage at week 4 (p = 0.024) and week 7 (p = 0.029), and median CFUs at week 7 (p = 0.050)., Conclusions: Fluconazole significantly reduces RIOM severity and Candida colonization in HNC patients undergoing CCRT, suggesting its efficacy as a prophylactic treatment., (© 2024 The Author(s). Head & Neck published by Wiley Periodicals LLC.)

The adverse outcomes of fungal infection in mammalian hosts depend on the complex interactions between the host immune system and pathogen virulence-associated traits. The main clinical problems arise when the host response is either too weak to effectively eliminate the pathogen or overly aggressive, resulting in host tissue damage rather than protection. This article will highlight current knowledge regarding the virulence attributions and mechanisms involved in the dual-sided role of the host immune system in the immunopathogenesis of the thermally dimorphic fungus Talaromyces marneffei through the lens of the damage response framework (DRF) of microbial pathogenesis model., 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 © 2024 Pruksaphon, Amsri, Jeenkeawpieam, Thammasit, Nosanchuk and Youngchim.)

Objective: Cryptococcosis predominantly presents as a meningoencephalitis in Thailand. Early and expeditious diagnosis is essential for reducing both mortality and morbidity associated with cryptococcal meningitis. We aim to define and establish the diagnostic performances between the benchmark commercially available diagnostic kit (CrAg® LFA) and the large-scale prototype of an inexpensive in-house immunochromatographic test (ICT) based on monoclonal antibody (MAb) 18B7., Methods: We have developed the large-scale prototype for the rapid detection of cryptococcal polysaccharide antigens by utilizing a single antibody sandwich ICT format employing MAb 18B7, which is highly specific to Cryptococcus neoformans glucuronoxylomannan (GXM) antigens. An in-house MAb18B7 ICT was manufactured in accordance with industry standards under the control of the International Organization for Standardization (ISO) 13485., Results: The diagnostic sensitivity, specificity, and accuracy for the in-house MAb 18B7 ICT were 99.10%, 97.61%, and 97.83%, respectively. The agreement kappa (κ) coefficient was 0.968 based on the retrospective evaluation of 580 specimens from patients living in northern Thailand with clinically suspected cryptococcosis., Conclusion: The data suggest that this in-house MAb 18B7 ICT will be highly beneficial for addressing the issue of cryptococcal infection in Thailand. Moreover, it is anticipated that this inexpensive ICT can play a pivotal role in various global strategies aimed at eradicating cryptococcal meningitis among individuals living with HIV by 2030., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic predators suggests that the pressure from predatory selection can significantly influence the development of fungal virulence and evolutionary processes. Thus, the purpose of this study was to investigate the adaptation of saprotrophic Candida albicans strains during their interactions with Acanthamoeba castellanii . We conducted a comprehensive analysis of survival after co-culture by colony counting of the yeast cells and examining yeast cell phenotypic and genetic characteristics. Our results indicated that exposure to amoebae enhanced the survival capacity of environmental C. albicans and induced visible morphological alterations in C. albicans , particularly by an increase in filamentation. These observed phenotypic changes were closely related to concurrent genetic variations. Notably, mutations in genes encoding transcriptional repressors ( TUP1 and SSN6 ), recognized for their negative regulation of filamentous growth, were exclusively identified in amoeba-passaged isolates, and absent in unexposed isolates. Furthermore, these adaptations increased the exposed isolates' fitness against various stressors, simultaneously enhancing virulence factors and demonstrating an increased ability to invade A549 lung human epithelial cells. These observations indicate that the sustained survival of C. albicans under ongoing amoebic predation involved a key role of mutation events in microevolution to modulate the ability of these isolates to change phenotype and increase their virulence factors, demonstrating an enhanced potential to survive in diverse environmental niches., 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 © 2024 Amsri, Pruksaphon, Thammasit, Nosanchuk and Youngchim.)

Talaromyces ( Penicillium ) marneffei (TM) is an important, but neglected, thermally dimorphic fungus. It is the pathogenic cause of talaromycosis, which is strongly associated with the immunodeficiency state present in individuals with advanced HIV disease. The purpose of this study was to develop a sandwich enzyme-linked immunosorbent assay (sandwich ELISA) for the detection of T . marneffei cytoplasmic yeast antigen (TM CYA) in human urine. Monoclonal antibody (MAb) 4D1 specifically binds to TM CYA. Galanthus nivalis agglutinin (GNA), a mannose -binding lectin, recognizes and binds to mannose residues of TM CYA. For the sandwich ELISA, the microplate was coated with GNA as the capturing molecule for absorbing immune complexes of MAb 4D1-TM CYA. The MAb 4D1-GNA sandwich ELISA did not detect a cross-reaction with other antigens from other fungi or bacteria. Seventy-four urine samples from patients with blood culture -confirmed talaromycosis and 229 urine samples from people without talaromycosis residing in the endemic area were subjected to the MAb 4D1-GNA sandwich ELISA. At an optical density (OD) cutoff value of 0.356, the sensitivity was 89.19% [95% confidence interval (CI): 79.80% -95.22%]; the specificity was 98.69% (95% CI: 96.22% -99.73%). The diagnostic performance of the MAb 4D1-GNA sandwich ELISA was highly consistent with those of blood culture and the Platelia Aspergillus galactomannan (GM) ELISA kit. Collectively, the MAb 4D1-GNA sandwich ELISA is a promising technique for the rapid diagnosis of T . marneffei infection, which would facilitate the early treatment of patients with talaromycosis and it may be used to monitor treatment responses., 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 Shu, Pruksaphon, Nosanchuk, Thammasit and Youngchim.)

Pathogenic eukaryotes including fungi release extracellular vesicles (EVs) which are composed of a variety of bioactive components, including peptides, nucleic acids, polysaccharides, and membrane lipids. EVs contain virulence-associated molecules suggesting a crucial role of these structures in disease pathogenesis. EVs derived from the pathogenic yeast phase of Talaromyces (Penicillium) marneffei , a causative agent of systemic opportunistic mycoses "talaromycosis," were studied for their immunogenic components and immunomodulatory properties. Some important virulence factors in EVs including fungal melanin and yeast phase specific mannoprotein were determined by immunoblotting. Furthermore, fluorescence microscopy revealed that T. marneffei EVs were internalized by THP-1 human macrophages. Co-incubation of T. marneffei EVs with THP-1 human macrophages resulted in increased levels of supernatant interleukin (IL)-1β, IL-6 and IL-10. The expression of THP-1 macrophage surface CD86 was significantly increased after exposed to T. marneffei EVs. These findings support the hypothesis that fungal EVs play an important role in macrophage "classical" M1 polarization. T. marneffei EVs preparations also increased phagocytosis, suggesting that EV components stimulate THP-1 macrophages to produce effective antimicrobial compounds. In addition, T. marneffei EVs stimulated THP-1 macrophages were more effective at killing T. marneffei conidia. These results indicate that T. marneffei EVs can potently modulate macrophage functions, resulting in the activation of these innate immune cells to enhance their antimicrobial activity., 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 Pruksaphon, Amsri, Thammasit, Nosanchuk and Youngchim.)

Background: Malassezia furfur is a member of the human skin microbiomes that can cause various skin diseases. Dimorphism plays a role as the yeast phase predominates during skin colonisation whereas mycelial forms are observed in the scales of patients with pityriasis versicolor (PV). However, due to their condition-dependence for growth, it is difficult to culture M. furfur and this is an additional challenge for studying the pathogenicity of this fungus., Objective: To describe different media suitable for culturing Malassezia from the yeast phase into mycelial forms, with a particular focus on nutritional supplements and pH conditions., Methods: Clinical M. furfur isolates from patients with PV and healthy individuals were used to investigate Malassezia dimorphism as well as the activity and expression of lipase enzymes., Results: Our experimental media were significantly more likely to promote mycelial growth in strains from healthy individuals compared to those from patients with PV. Lipase activity was increased in the mycelial phase cells compared to yeast forms for all strains tested. Assessment of the relative transcriptional expression of lipase within M. furfur revealed that LIP-coding genes were upregulated in mycelium relative to yeast forms for the strains tested. However, the increases in LIP3, LIP5 and LIP6 gene expressions were significantly greater in strains from healthy individuals compared to those from patients with PV., Conclusion: Overall, this study validated effective growth conditions to study M. furfur virulence factors and demonstrated that lipase is associated with M. furfur dimorphism., (© 2023 Wiley-VCH GmbH.)

Talaromyces marneffei, a dimorphic fungus, exhibits temperature-dependent growth, existing in a filamentous form at 25 °C and as a yeast at 37 °C. Several studies have highlighted the important roles of macrophages in defense against T. marneffei infection. However, the immune responses to the interaction of macrophages with T. marneffei cells during phase transition require further investigation. This study reports the expression of cytokine profiles in human THP-1 cells during infection by T. marneffei. THP-1 cells were infected with T. marneffei conidia at different multiplicity of infections (MOIs). Surviving conidia transformed into yeasts after phagocytosis by macrophages, and the number of yeasts gradually increased over 36 h. The transcription and secretion levels of pro- and anti-inflammatory cytokines were examined at different times by qRT-PCR and ELISA. Transcription levels of IL-8, IL-12, IL-1β, and TNF-α increased significantly at 12 or 24 h and then slightly decreased at 36 h. In contrast, the transcription levels of IL-6, IL-10, and TGF-β gradually increased at all MOIs. The levels of IL-6 and IL-10 secretion corresponded to their levels of transcription. These results indicated that as the number of intracellular yeasts increased, the infected macrophages first underwent slight M1 polarization before shifting to M2 polarization. This polarization transition was confirmed by the fungicidal ability and the expression of macrophage surface markers. By inducing the M2-type polarization of macrophages, the intracellular T. marneffei cells can successfully evade the immune response. Our study provides a novel insight into the immune characterization during the transition of T. marneffei infection and could further contribute to possible diagnostic and therapeutic interventions for this infection., Competing Interests: The authors declare no conflict of interest.

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.)

Talaromycosis (Penicilliosis) is an opportunistic mycosis caused by the thermally dimorphic fungus Talaromyces ( Penicillium ) marneffei . Similar to other major causes of systemic mycoses, the extent of disease and outcomes are the results of complex interactions between this opportunistic human pathogen and a host's immune response. This review will highlight the current knowledge regarding the dynamic interaction between T. marneffei and mammalian hosts, particularly highlighting important aspects of virulence factors, intracellular lifestyle and the mechanisms of immune defense as well as the strategies of the pathogen for manipulating and evading host immune cells.

Talaromycosis (penicilliosis) is an invasive mycosis that is endemic in tropical and subtropical Asia. Talaromycosis primarily affects individuals with advanced HIV disease and other immunosuppressive conditions, and the disease disproportionally affects people in low-income and middle-income countries, particularly agricultural workers in rural areas during their most economically productive years. Approximately 17 300 talaromycosis cases and 4900 associated deaths occur annually. Talaromycosis is highly associated with the tropical monsoon season, when flooding and cyclones can exacerbate the poverty-inducing potential of the disease. Talaromycosis can present as localised or disseminated disease, the latter causing cutaneous lesions that are disfiguring and stigmatising. Despite up to a third of diagnosed cases resulting in death, talaromycosis has received little attention and investment from regional and global funders, policy makers, researchers, and industry. Diagnostic and treatment modalities remain extremely insufficient, however control of talaromycosis is feasible with known public health strategies. This Viewpoint is a global call for talaromycosis to be recognised as a neglected tropical disease to alleviate its impact on susceptible populations., Competing Interests: Declaration of interests NPG reports grants from US National Institutes of Health, US Centers for Disease Control and Prevention, the CDC Foundation, the Bill & Melinda Gates Foundation, the UK Medical Research Council, and the South African National Health Laboratory Service Research Trust, outside the submitted work. JF-WC and K-YY have an issued patent on antibodies targeting T marneffei Mp1p proteins and their methods of use. JP reports grants from Merck, Astellas, Pfizer, Amplyx, and Minnetronix; and membership on advisory boards or consulting roles for Merck, Amplyx, Minnetronix, F2G, Scynexis, Ampili, and Matinas, outside the submitted work. TL reports research funding from Gilead Sciences, outside the submitted work. All other authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Invasive pulmonary aspergillosis is a frequent complication in immunocompromised individuals, and it continues to be an important cause of mortality in patients undergoing hematopoietic stem cell transplantation. In addition to antifungal therapy used for mycoses, immune-modulatory molecules such as cytokines and chemokines can modify the host immune response and exhibit a promising form of antimicrobial therapeutics to combat invasive fungal diseases. Cytokine and chemokine profiles may also be applied as biomarkers during fungal infections and clinical research has demonstrated different activation patterns of cytokines in invasive mycoses such as aspergillosis. In this review, we summarize different aspects of cytokines that have been described to date and provide possible future directions in research on invasive pulmonary aspergillosis following hematopoietic stem cell transplantation. These findings suggest that cytokines and chemokines may serve as useful biomarkers to improve diagnosis and monitoring of infection.

Fungal keratitis (FK) is a serious ocular infection that can result in various degrees of vision loss, including blindness. The aim of the study was to identify and retrospectively review all FK cases diagnosed between August 2012 and December 2020 at a tertiary care hospital in northern Thailand with a specific focus on epidemiologic features, including season, patient sex and age, the spectrum of pathogens, and presence of certain putative virulence factors. Of 1237 patients with corneal ulcers, 294 (23.8%) were confirmed by direct microscopic examination and/or fungal culture. For the positive cases, direct examinations of Calcofluor white (CW) stains and KOH mounts were found in 97.3% (286/294) and 76.5% (225/294), respectively ( p < 0.05). Of the cases diagnosed by microscopy and culture, fungi were isolated in 152 (51.7%), with Fusarium spp. being the most frequently identified ( n = 69, 45.5%) followed by dematiaceous fungi ( n = 45, 29.6%) and Aspergillus spp. ( n = 18, 11.8%). The incidence of FK was higher in the rainy season of July to October. The mean age was 54.4 ± 14.4 (SD) years, with a range of 9-88 years. Males (75.8%) were affected significantly more than females (24.2%) ( p < 0.05). Of 294 patients, 132 (44.9%) were middle-aged adults (41-60 years) and 107 (36.4%) were older than 60 years. Trauma to the eye by soil or vegetative matter were the most common preceding factors (188/294; 64.0%). We assessed two virulence factors. First, 142 of the 152 culture-positive FK cases were due to molds, indicating that hyphal morphogenesis is extremely important in disease. We also demonstrated that fungal melanization occurs in the molds during the course of FK by applying a melanin-specific monoclonal antibody (MAb) that labeled fungal elements in corneal samples of patients, and melanin particles derived from the hyphae were also recovered after treatment of the samples with proteolytic enzymes, denaturant and hot concentrated acid. In summary, we demonstrate that northern Thailand has a high rate of FK that is influenced by season and males engaged in outside activities are at highest risk for disease. Moulds are significantly more commonly responsible for FK, in part due to their capacity to form hyphae and melanins. Future studies will examine models of fungal corneal interactions and assess additional factors of virulence, such as secreted enzymes, to more deeply decipher the pathogenesis of FK.

Talaromyces marneffei is a thermally dimorphic fungus that causes opportunistic systemic mycoses in patients with AIDS or other immunodeficiency syndromes. The purpose of this study was to develop an immunochromatographic strip test (ICT) based on a solid phase sandwich format immunoassay for the detection of T. marneffei antigens in clinical urine specimens. The T. marneffei yeast phase specific monoclonal antibody 4D1 (MAb4D1) conjugated with colloidal gold nanoparticle was used as a specific signal reporter. Galanthus nivalis Agglutinin (GNA) was adsorbed onto nitrocellulose membrane to serve as the test line. Similarly, a control line was created above the test line by immobilization of rabbit anti-mouse IgG. The immobilized GNA served as capturing molecule and as non-immune mediated anti-terminal mannose of T. marneffei antigenic mannoprotein. The MAb4D1-GNA based ICT showed specific binding activity with yeast phase antigen of T. marneffei, and it did not react with other common pathogenic fungal antigens. The limit of detection of this ICT for T. marneffei antigen spiked in normal urine was approximately 0.6 μg/ml. The diagnostic performance of the ICT was validated using 341 urine samples from patents with culture- confirmed T. marneffei infection and from a control group of healthy individuals and patients with other infections in an endemic area. The ICT exhibited 89.47% sensitivity, 100% specificity, and 97.65% accuracy. Our results demonstrate that the urine-based GNA-MAb4D1 based ICT produces a visual result within 30 minutes and that the test is highly specific for the diagnosis of T. marneffei infection. The findings validate the deployment of the ICT for clinical use., Competing Interests: The authors have declared that no competing interests exist.

The aim of this study was to develop a novel lateral flow immunochromatoghaphic strip test (ICT) for detecting cryptococcal polysaccharide capsular antigens using only a single specific monoclonal antibody, mAb 18B7. The mAb 18B7 is a well characterized antibody that specifically binds repeating epitopes displayed on the cryptococcal polysaccharide glucuronoxylomannan (GXM). We validated the immunoreactivities of mAb 18B7 against capsular antigens of different cryptococcal serotypes. The mAb 18B7 ICT was constructed as a sandwich ICT strip and the antibody serving in the mobile phase (colloidal gold conjugated mAb 18B7) to bind one of the GXM epitopes while the stationary phase antibody (immobilized mAb18B7 on test line) binding to other remaining unoccupied epitopes to generate a positive visual readout. The lower limit of detection of capsular antigens for each of the Cryptococcus serotypes tested was 0.63 ng/mL. No cross-reaction was found against a panel of antigens isolated from cultures of other pathogenic fungal, except the crude antigen of Trichosporon sp. with the lower limit of detection of 500 ng/mL (~800 times higher than that for cryptococcal GXM). The performance of the mAb 18B7 ICT strip was studied using cerebrospinal fluid (CSF) and serum and compared to commercial diagnostic kits (latex agglutination CALAS and CrAg IMMY). The sensitivity, specificity and accuracy of the mAb18B7 ICT with CSF from patients with confirmed cryptococcal meningitis were 92.86%, 100% and 96.23%, respectively. No false positives were observed with samples from non-cryptococcosis patients. With serum samples, the mAb 18B7 ICT gave a sensitivity, specificity and accuracy of 96.15%, 97.78% and 96.91%, respectively. Our results show that the mAb 18B7 based ICT was reliable, reproducible, and cost-effective as a point-of-care immunodiagnostic test for cryptococcosis. The mAb 18B7 ICT may be particularly useful in countries where commercial kits are not available or affordable.

Melanins are ubiquitous complex polymers that are commonly known in humans to cause pigmentation of our skin. Melanins are also present in bacteria, fungi, and helminths. In this review, we will describe the diverse interactions of fungal melanin with the mammalian immune system. We will particularly focus on Cryptococcus neoformans and also discuss other major melanotic pathogenic fungi. Melanin interacts with the immune system through diverse pathways, reducing the effectiveness of phagocytic cells, binding effector molecules and antifungals, and modifying complement and antibody responses.

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.

The pathogenic fungus Talaromyces (formerly Penicillium) marneffei is a thermally dimorphic fungus that can cause disseminated infection in patients with secondary immunodeficiency syndrome, in particular in the setting of advanced HIV infection. The areas of highest incidence are in Southeast Asia, Southern China, and Indian subcontinents. Talaromycosis (formerly penicilliosis) is identified as an AIDS-defining illness, and it has recently been recognized in non-HIV-associated patients with impaired cellular-mediated immunity. Microbiological culture is the gold standard method for the diagnosis of T. marneffei infection and usually requires up to 2-4 weeks for detectable growth to occur, which may result in a delay of appropriate treatment. Immunodiagnosis has become an alternative method for confirming talaromycosis. This article reviews various immunological tests for the diagnosis of talaromycosis, including a proposed novel rapid point-of-care assay using a new T. marneffei yeast phase-specific monoclonal antibody., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Melanins are one of the great natural pigments produced by a wide variety of fungal species that promote fitness and cell survival in diverse hostile environments, including during mammalian infection. In this study, we sought to demonstrate the production of melanin in the conidia and hyphae of saprophytic fungi, including dematiaceous and hyaline fungi. We showed that a melanin-specific monoclonal antibody (MAb) avidly labeled the cell walls of hyphae and conidia, consistent with the presence of melanin in these structures, in 14 diverse fungal species. The conidia of saprophytic fungi were treated with proteolytic enzymes, denaturant, and concentrated hot acid to yield dark particles, which were shown to be stable free radicals, consistent with their identification as melanins. Samples obtained from patients with fungal keratitis due to Fusarium falciforme, Aspergillus fumigatus, Aspergillus flavus, Curvularia lunata, Exserohilum rostratum, or Fonsecaea pedrosoi were found to be intensely labeled by the melanin-specific MAb at the fungal hyphal cell walls. These results support the hypothesis that melanin is a common component that promotes survival under harsh conditions and facilitates fungal virulence. Increased understanding of the processes of melanization and the development of methods to interfere with pigment formation may lead to novel approaches to combat these complex pathogens that are associated with high rates of morbidity and mortality., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Talaromyces (Penicillium) marneffei is a thermally dimorphic fungus that can cause opportunistic systemic mycoses in patients infected with the human immunodeficiency virus (HIV). It has also been reported among patients with other causes of immunodeficiency, such as systemic lupus erythematosus, cancer, organ transplanted patients receiving immunosuppressive drug and adult onset immunodeficiency syndromes. Recent studies indicate that the clinical manifestations, laboratory findings and treatment strategies of talaromycosis (penicilliosis) marneffei are different between patients with and without HIV infection. Therefore early and accurate diagnosis of talaromycosis marneffei is crucial to the proper management and treatment. Since current diagnostic methods are currently inadequate, the aim of this study was to develop an immunochromatographic test (ICT) for the detection of T. marneffei yeast antigens in urine samples. The highly T. marneffei-specific monoclonal antibody 4D1 (MAb 4D1) conjugated with gold colloid at pH 6.5 was used as signal generator. The nitrocellulose membrane was lined with T. marneffei cytoplasmic yeast antigen (TM CYA) to serve as the test line, and rabbit anti-mouse IgG was the control line. Subjecting the assembled test strip to urine samples containing T. marneffei antigen produced a visible result within 20 minutes. The sensitivity limit of the assay was 3.125μg/ml of TM CYA. The ICT was used to test urine samples from 66 patients with blood culture confirmed talaromycosis marneffei, 42 patients with other fungal or bacterial infections, and 70 normal healthy individuals from endemic area of T. marneffei. The test exhibited sensitivity, specificity and accuracy of 87.87%, 100% and 95.5%, respectively. This rapid, user-friendly test holds great promise for the serodiagnosis of T. marneffei infection.

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.

Snake envenomation is an important medical problem. One of the hurdles in antivenom development is the in vivo assay of antivenom potency which is expensive, gives variable results and kills many animals. We report a novel in vitro assay involving the specific binding of the postsynaptic neurotoxins (PSNTs) of elapid snakes with purified Torpedo californica nicotinic acetylcholine receptor (nAChR). The potency of an antivenom is determined by its antibody ability to bind and neutralize the PSNT, thus preventing it from binding to nAChR. The PSNT of Naja kaouthia (NK3) was immobilized on microtiter wells and nAChR was added to bind with it. The in vitro IC 50 of N. kaouthia venom that inhibited 50% of nAChR binding to the immobilized NK3 was determined. Varying concentrations of antisera against N. kaouthia were separately pre-incubated with 5xIC 50 of N. kaouthia venom. The remaining free NK3 were incubated with nAChR before adding to the NK3 coated plates. The in vitro and in vivo median effective ratio, ER 50 s of 12 batches of antisera showed correlation (R 2 ) of 0.9809 (p < 0.0001). This in vitro assay should be applicable to antisera against other elapid venoms and should reduce the use of live animals and accelerate development of life-saving antivenoms.

Malassezia furfur, a constituent of the normal human skin flora, is an etiological agent of pityriasis versicolor, which represents one of the most common human skin diseases. Under certain conditions, both exogenous and endogenous, the fungus can transition from a yeast form to a pathogenic mycelial form. To develop a standardized medium for reproducible production of the mycelial form of M. furfur to develop and optimize susceptibility testing for this pathogen, we examined and characterized variables, including kojic acid and glycine concentration, agar percentage, and pH, to generate a chemically defined minimal medium on which specific inoculums of M. furfur generated the most robust filamentation. Next, we examined the capacity of ketoconazole to inhibit the formation of M. furfur mycelial form. Both low and high, 0.01, 0.05 and 0.1 µg/ml concentrations of ketoconazole significantly inhibited filamentation at 11.9, 54.5 and 86.7%, respectively. Although ketoconazole can have a direct antifungal effect on both M. furfur yeast and mycelial cells, ketoconazole also has a dramatic impact on suppressing morphogenesis. Since mycelia typified the pathogenic form of Malassezia infection, the capacity of ketoconazole to block morphogenesis may represent an additional important effect of the antifungal.

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 (Penicillium) marneffei is an emerging opportunistic pathogen associated with HIV infection, particularly in Southeast Asia and southern China. The rapid uptake and killing of T. marneffei conidia by phagocytic cells along with the effective induction of an inflammatory response by the host is essential for disease control. T. marneffei produces a number of different laccases linked to fungal virulence. To understand the role of the various laccases in T. marneffei, laccase-encoding genes were investigated. Targeted single, double and triple gene deletions of laccases encoding lacA, lacB, and lacC showed no significant phenotypic effects suggesting redundancy of function. When a fourth laccase-encoding gene, pbrB, was deleted in the ΔlacA ΔlacB ΔlacC background, the quadruple mutant displayed delayed conidiation and the conidia were more sensitive to H2O2, sodium dodecyl sulfate (SDS), and antifungal agents than wild-type and other transformants. Conidia of the quadruple mutant showed marked differences in their interaction with the human monocyte cell line, THP-1 such that phagocytosis was significantly higher when compared with the wild-type at one and 2 hours of incubation while the phagocytic index was significantly different from 15 to 120 minutes. In addition, killing of the quadruple mutant by THP-1 cells was more efficient at 2 and 4 hours of incubation. The levels of the proinflammatory cytokines TNF-α, IL-1β and IL-6 from THP-1 cells infected with the quadruple mutant were also significantly increased in comparison with wild-type. The results demonstrate that production of laccases by T. marneffei actually promotes the pathogen's resistance to innate host defenses.

The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment.

Penicillium marneffei, the pathogenic thermal dimorphic fungus is a causative agent of a fatal systemic disease, penicilliosis marneffei, in immunocompromised patients especially HIV patients. For growth and survival, this fungus has to adapt to environmental stresses outside and inside host cells and this adaptation requires stress signaling pathways and regulation of gene expression under various kinds of stresses. In this report, P. marneffei activating transcription factor (atfA) gene encoding bZip-type transcription factor was characterized. To determine functions of this gene, atfA isogenic mutant strain was constructed using the modified split marker recombination method. The phenotypes and susceptibility to varieties of stresses including osmotic, oxidative, heat, UV, cell wall and cell membrane stresses of the mutant strain were compared with the wild type and the atfA complemented strains. Results demonstrated that the mRNA expression level of P. marneffei atfA gene increased under heat stress at 42°C. The atfA mutant was more sensitive to sodium dodecyl sulphate, amphotericin B and tert-butyl hydroperoxide than the wild type and complemented strains but not hydrogen peroxide, menadione, NaCl, sorbitol, calcofluor white, itraconazole, UV stresses and heat stress at 39°C. In addition, recovery of atfA mutant conidia after mouse and human macrophage infections was significantly decreased compared to those of wild type and complemented strains. These results indicated that the atfA gene was required by P. marneffei under specific stress conditions and might be necessary for fighting against host immune cells during the initiation of infection.

The biosynthesis of melanin has been linked with virulence in diverse pathogenic fungi. Penicillium marneffei, a dimorphic fungus, is capable of melanization in both mycelial and yeast phases, and the pigment may be produced during infection to protect the fungus from the host immune system. To investigate the impact of yeast morphological transformation on antifungal susceptibility, P. marneffei was cultured on various media including minimal medium, 1 % tryptone, brain heart infusion broth, and malt extract broth by using the standardized susceptibility protocol (the M27-A protocol, RPMI medium) for yeasts. We also investigated whether P. marneffei melanization affected its susceptibility to antifungal drugs by adding L-DOPA into culture broths. There were no differences in the minimum inhibitory concentrations of P. marneffei yeast cells previously grown in various culture broths with or without L-DOPA using the M27A protocol (into which no melanin substrate can be added due to a rapid colour change of the RPMI medium to black) for testing amphotericin B, clotrimazole, fluconazole, itraconazole and ketoconazole. However, both melanized and non-melanized P. marneffei displayed increased resistance to antifungal drugs when L-DOPA was added into a selected assay medium, 0.17 % yeast nitrogen base, 2 % glucose, and 1.5 % agar. Hence, active melanin formation appears to protect P. marneffei by enhancing its resistance to antifungal drugs.

Background: Infections caused by Malassezia yeasts are most likely underdiagnosed, because fatty acid supplementation is needed for growth. Rapid identification of Malassezia species is essential for appropriate treatment of Malassezia-related skin infections, fungaemia and nosocomial outbreaks in neonates, children and adults and can be life-saving for those patients. Ma-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been reported to be a rapid and reliable diagnostic tool to identify clinically important yeasts, but so far no data have been reported on identification of Malassezia isolates with this technique., Objectives: To create an extensive database of main mass spectra (MSPs) that will allow quick identification of Malassezia species by MALDI-TOF MS., Methods: An in-house library of 113 MSPs was created from 48 reference strains from the CBS-KNAW yeast collection. The in-house library was challenged with two test sets of Malassezia strains, namely 165 reference strains from the CBS collection and 338 isolates collected in Greece, Italy, Sweden and Thailand., Results: MALDI-TOF MS allowed correct identification of all 14 Malassezia spp. MALDI-TOF MS results were concordant with those of sequence analyses of the internal transcribed spacers (ITS1/ITS2) and the D1/D2 domains of the large subunit of the ribosomal DNA., Conclusions: Implementation of the MALDI-TOF MS system as a routine identification tool will contribute to correct identification of Malassezia yeasts with minimal effort and in a short turnaround time, which is especially important for the rapid identification of Malassezia in skin diseases and nosocomial outbreaks., (© 2013 British Association of Dermatologists.)

Melanins are synthesized by organisms of all biological kingdoms and comprise a heterogeneous class of natural pigments. Certain of these polymers have been implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the fungal skin pathogen Malassezia furfur produces melanin or melanin-like compounds. A melanin-binding monoclonal antibody (MAb) labelled in vitro cultivated yeast cells of M. furfur. In addition, melanization of Malassezia yeasts and hyphae was detected by anti-melanin MAb in scrapings from patients with pityriasis versicolor. Treatment of Malassezia yeasts with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles and electron spin resonance spectroscopy revealed that these particles contained a stable free radical compound, consistent with their identification as melanins. Malassezia yeasts required phenolic compounds, such as L-DOPA, in order to synthesize melanin. L-DOPA also triggered hyphal formation in vitro when combined with kojic acid, a tyrosinase inhibitor, in a dose-dependent manner. In this respect, L-DOPA is thought to be an essential substance that is linked to both melanization and yeast-mycelial transformation in M. furfur. In summary, M. furfur can produce melanin or melanin-like compounds in vitro and in vivo, and the DOPA melanin pathway is involved in cell wall melanization.

Dermatophytes are keratinophilic fungi that are the most common cause of fungal skin infections worldwide. Melanin has been isolated from several important human fungal pathogens, and the polymeric pigment is now recognized as an important virulence determinant. This study investigated whether dermatophytes, including Trichophyton rubrum, Trichophyton mentagrophytes, Epidermophyton floccosum and Microsporum gypseum, produce melanin or melanin-like compounds in vitro and during infection. Digestion of the pigmented microconidia and macroconidia of dermatophytes with proteolytic enzymes, denaturant and hot concentrated acid yielded dark particles that retained the size and shape of the original fungal cells. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical signal, consistent with the pigments being a melanin. Immunofluorescence analysis demonstrated reactivity of a melanin-binding mAb with the pigmented conidia and hyphae, as well as the isolate particles. Laccase, an enzyme involved in melanization, was detected in the dermatophytes by an agar plate assay using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. Skin scrapings from patients with dermatophytoses contained septate hyphae and arthrospores that were reactive with the melanin-binding mAb. These findings indicate that dermatophytes can produce melanin or melanin-like compounds in vitro and during infection. Based on what is known about the function of melanin as a virulence factor of other pathogenic fungi, this pigment may have a similar role in the pathogenesis of dermatophytic diseases.

Melanins are found universally in nature and are implicated in the pathogenesis of several important human fungal pathogens. This study investigated whether the conidia and the yeast cells of the thermally dimorphic fungal pathogen Penicillium marneffei produce melanin or melanin-like compounds in vitro and during infection. Treatment of conidia with proteolytic enzymes, denaturant and concentrated hot acid yielded dark particles that were similar in size and shape to the conidia. A melanin-binding monoclonal antibody (mAb) labelled pigmented conidia, yeast cells and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that particles derived from pigmented conidia contained a stable free radical compound, consistent with their identification as melanins. Skin tissue from penicilliosis marneffei patients contained yeast cells that were labelled by melanin-binding mAb. Additionally, sera from P. marneffei-infected mice developed a significant antibody response (both IgG and IgM) against melanin. Phenoloxidase activity capable of synthesizing melanin from l-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that P. marneffei conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that the yeast cells can synthesize pigment in vivo. Accordingly this pigment may play some role in the virulence of P. marneffei.

Scytalidium dimidiatum is a pigmented dematiaceous coelomycete that typically causes chronic superficial skin diseases and onychomycosis, as well as deeper infections, such as subcutaneous abscesses, mycetoma, and even fungemia in immunocompromised patients. A second species, Scytalidium hyalinum, has hyaline hyphae and arthroconidia and is considered by some authors to be an albino mutant of S. dimidiatum. This study aimed to confirm the presence of melanin or melanin-like compounds (which have been previously implicated in the virulence of other fungal pathogens) in S. dimidiatum from a patient with multiple subcutaneous nodules. Treatment of the hyphae and arthroconidia with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles, which were stable free radicals, consistent with their identification as melanins. Extracted melanin particles from S. dimidiatum cultures were labeled by melanin-binding monoclonal antibodies (MAbs) from Sporothrix schenckii, Aspergillus fumigatus, and Cryptococcus neoformans. Lesional skin from the patient infected with S. dimidiatum contained fungal cells that were labeled by melanin-binding MAbs, and digestion of the tissue yielded dark particles that were also reactive. S. hyalinum was also subjected to the melanin extraction protocol, but no dark particles were yielded.

Melanins, or melanin-like compounds, may play a role in the pathogenesis of a number of human fungal infections. This study investigated the production of melanin by the important opportunistic pathogen Aspergillus fumigatus. Conidia from A. fumigatus were harvested and treated with proteolytic enzymes, denaturant and hot, concentrated acid; this yielded dark particles which were similar in size and shape to the original propagules. Electron spin resonance spectroscopy revealed that the conidial-derived particles were stable free radicals consistent with an identification as melanin. Melanin particles were used to immunize BALB/c mice in order to produce a total of five anti-melanin monoclonal antibodies (mAbs). The latter mAbs were strongly reactive both with intact conidia and with extracted melanin particles by ELISA and immunofluorescence reactivity. Immunofluorescence labelling with the novel mAbs was used to examine the temporal expression of melanin during in vitro culture of A. fumigatus--melanization was confined to conidial structures and was absent from hyphae. SDS-PAGE L-3,4-dihydroxyphenylalanine (L-DOPA) substrate analysis confirmed the presence of a laccase-type activity in conidial extracts, but not in hyphae. Melanin-binding mAbs were used to detect the presence of melanized conidia in three patients with nasal aspergilloma, indicating that in vivo melanization may occur during infection.

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with L-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

Melanins are implicated in the pathogenesis of several human diseases, including some microbial infections. In this study, we analyzed whether the conidia and the yeasts of the thermally dimorphic fungal pathogen Paracoccidioides brasiliensis produce melanin or melanin-like compounds in vitro and during infection. Growth of P. brasiliensis mycelia on water agar alone produced pigmented conidia, and growth of yeasts in minimal medium with L-3,4-dihydroxyphenylalanine (L-DOPA) produced pigmented cells. Digestion of the pigmented conidia and yeasts with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were the same size and shape as their propagules. Immunofluorescence analysis demonstrated reactivity of a melanin-binding monoclonal antibody (MAb) with the pigmented conidia, yeasts, and particles. Electron spin resonance spectroscopy identified the yeast-derived particles produced in vitro when P. brasiliensis was grown in L-DOPA medium as a melanin-like compound. Nonreducing polyacrylamide gel electrophoresis of cytoplasmic yeast extract revealed a protein that catalyzed melanin synthesis from L-DOPA. The melanin binding MAb reacted with yeast cells in tissue from mice infected with P. brasiliensis. Finally digestion of infected tissue liberated particles reactive to the melanin binding MAb that had the typical morphology of P. brasiliensis yeasts. These data strongly suggest that P. brasiliensis propagules, both conidia and yeast cells, can produce melanin or melanin-like compounds in vitro and in vivo. Based on what is known about the function of melanin in the virulence of other fungi, this pigment may play a role in the pathogenesis of paracoccidioidomycosis.

The cell-associated and extracellular enzymatic activities were examined in a total of 10 Penicillium marneffei isolates. Both mycelia and yeast expressed alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase activities, whereas a variety of other enzyme activities, including trypsin, chymotrypsin and alpha-fucosidase were absent. There was some inter-isolate variation in both mycelia and yeast in the activities of other enzymes such as esterases and galactosidases. Enzyme activities did not change significantly over the course of culturing in three representative isolates.

Adhesion of Penicillium marneffei conidia to the extracellular matrix protein laminin via a sialic acid-dependent process has previously been demonstrated. This study describes the interaction of P. marneffei conidia with fibronectin and examines the relationship of this process to the recognition of laminin via conidia. Immunofluorescence microscopy demonstrated that fibronectin bound to the surface of conidia and to phialides, but not to hyphae, in a pattern similar to that reported for laminin. Conidia were able to bind to fibronectin immobilized on microtiter plates in a concentration-dependent manner. However, binding to fibronectin (at any given concentration of protein and conidia) was less than that to laminin under equivalent conditions. Soluble fibronectin and antifibronectin antibody inhibited adherence of conidia to fibronectin in the plate adherence assay; soluble laminin also caused pronounced inhibition. Various monosaccharides and several peptides had no effect on adherence to fibronectin. However, N-acetylneuraminic acid abolished adherence to fibronectin, indicating that the interaction was mediated through a sialic acid-dependent process; the latter parallels observations of laminin binding by conidia. Fibronectin binding (and binding of laminin) was considerably reduced by prolonged preincubation of conidia with chymotrypsin, suggesting the protein nature of the binding site. Conidia from older cultures were more adherent to both immobilized fibronectin and laminin than conidia from younger cultures. Ligand affinity binding demonstrated the presence of a 20-kDa protein with the ability to bind both fibronectin and laminin. There would therefore appear to be a common receptor for the binding of fibronectin and laminin on the surface of P. marneffei, and the interaction described here maybe important in mediating attachment of the fungus to host tissue.

Immunofluorescence microscopy demonstrated that laminin bound to the surface of Penicillium marneffei conidia. Attachment of P. marneffei conidia in an adherence assay was inhibited by soluble laminin and anti-laminin antibody. N-Acetylneuraminic acid abolished adherence, indicating an interaction mediated by a sialic acid-specific lectin.

Forty-six isolates of Penicillium marneffei were differentiated into two DNA types on the basis of their restriction fragment length polymorphisms. Of the 22 human isolates of P. marneffei, 16 (72.7%) were type I and 6 (27.3%) were type II. Of the 23 bamboo rat isolates, 20 from Rhizomys sumatrensis were type I and 3 from Cannomys badius were type II. The soil isolate was type II. These data represent the first molecular epidemiological study of this important emerging fungal pathogen.