Adaptation to an amoeba host drives selection of virulence-associated traits and genetic variation in saprotrophic Candida albicans.

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.

Extracellular vesicles derived from Talaromyces marneffei contain immunogenic compounds and modulate THP-1 macrophage responses.

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)-1ß, 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.

Antibody screening reveals antigenic proteins involved in Talaromyces marneffei and human interaction.

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.

Genetic Engineering of Talaromyces marneffei to Enhance Siderophore Production and Preliminary Testing for Medical Application Potential.

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 (ΔsreA) produced a high amount of extracellular siderophores. The produced siderophore was characterized using HPLC-MS, HPLC-DAD, FTIR, and 1H- and 13C-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.

Role of acuK in Control of Iron Acquisition and Gluconeogenesis in Talaromyces marneffei.

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, ΔacuK showed double nuclei hyphae. However, the yeast cells showed normal morphology. The ΔacuK failed to grow in iron-limiting conditions. Additionally, it could not grow in a medium containing gluconeogenic carbon sources. Moreover, ΔacuK 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.

Expression of Talaromyces marneffei acuM and acuK Genes in Gluconeogenic Substrates and Various Iron Concentrations.

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.