Role of AcuK and AcuM transcription factors in dimorphic fungus Talaromyces marneffei: Analysis of target gene expression and macrophage interaction

To identify the role of acuK and acuM transcription factors in <i>Talaromyces marneffei</i>, we examined the ability of the <i>acuK</i> and <i>acuM</i> deletion mutants to (i) regulate target genes involved in iron metabolism and gluconeogenesis, (ii) produce siderophore, and (iii) interact with host macrophage THP1 cells.<b>Real-time PCR</b>: <i>T. marneffei</i> ATCC18224, Δ<i>acuK,</i> and Δ<i>acuM</i> strains were prepared to 10⁸ conidia/ml in 100 ml ANM medium and were pre-cultivated at 25 °C for 16 hours to generate germinating conidia. The fungal cells were washed three times with sterile PBS before being transferred into a new medium for different test conditions. For detection of the gluconeogenic genes, the pre-cultivated fungal cells were transferred to the glucose-free medium containing the following gluconeogenic carbon sources; 50 mM proline, 50 mM acetate, and 0.5% ethanol. To investigate the expression of genes in the iron assimilation pathways , the pre-culture was transferred to the iron-free ANM broth supplemented with different iron concentrations (0 – 1000 mM FeCl₃). The cultures were incubated at either 25 °C for 24 hours or 37 °C for 48 hours before harvesting the fungal cells.<b>Siderophore production</b>: To investigate the involvement of <i>acuK</i> and <i>acuM</i> in siderophore biosynthesis, the amount of siderophore production was measured in the wild type, ∆<i>acuK</i> and ∆<i>acuM </i>strains, using a Chrome Azurol S (CAS) solid and liquid assay. First, <i>T. marneffei</i> wild type and mutants were cultivated in ANM broth for 7 days. The amount of extracellular siderophores was measured from culture supernatant while the intracellular siderophores were determined from cell lysates.<b>Macrophage infection</b>: The THP-1 human monocytic cell line was used for a macrophage infection model, with cells maintained in RPMI 1640 medium (Life Technologies, Grand Island, USA) with 10% FBS (v/v) at 37 °C, 5% CO₂. THP-1 cells (1 × 10⁶) were inoculated into each per well of a 6-well microtiter plate containing one sterile coverslip for phagocytosis assay and a 12-well plate for killing assay. The cell was differentiated with 32 μM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for 24 h at 37 °C and 5% CO₂. Then, 1 × 10⁶ <i>T. marneffei</i> conidia of G809 (<i>acu</i><i>K</i>⁺<i>acuM</i>⁺), Δ<i>acuK</i>, and Δ<i>acuM</i> were added to the macrophages and infected for 2 h. For the phagocytosis assay, macrophages were fixed with 4% paraformaldehyde and stained with 1 mg/ml calcofluor white to observe the fungal cell walls. For the killing assay at 2 h after inoculation, the macrophage cells were harvested and lysed with 0.25% TritonX-100 (Sigma-Aldrich). The macrophage killing at 24 h was also determined. After 2 h of infection, the macrophage cells were washed three times with PBS, and the infection was continued for an additional 24 hours before cell lysis. The recovered fungal cells were plated on a synthetic dextrose agar (SD; 0.17% w/v yeast nitrogen base without amino acids, 2% w/v glucose, 10 mM (NH₄)₂SO₄, 2% agar). Colony forming units (CFU) were determined after growth at 37 °C for 7–10 days.