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.

为明确acuK与acuM转录因子在马尔尼菲篮状菌（Talaromyces marneffei）中的功能，我们检测了acuK和acuM基因缺失突变体的三项能力：(i) 调控铁代谢与糖异生相关靶基因，(ii) 合成铁载体，(iii) 与宿主THP1巨噬细胞相互作用。
实时荧光定量PCR（Real-time PCR）：将马尔尼菲篮状菌ATCC18224野生株、ΔacuK突变株与ΔacuM突变株制备为浓度10^8 分生孢子/mL的菌液，接种至100 mL ANM培养基中，于25 ℃预培养16小时以获得萌发的分生孢子。随后用无菌PBS洗涤真菌细胞三次，再转接至适配不同实验条件的新鲜培养基中。针对糖异生基因的检测，将预培养后的真菌细胞转接至不含葡萄糖且添加以下糖异生碳源的培养基：50 mM 脯氨酸、50 mM 乙酸钠与0.5%乙醇。为探究铁同化通路相关基因的表达，将预培养的真菌细胞转接至无铁ANM液体培养基中，并添加不同浓度梯度的FeCl3（0 – 1000 mM）。将培养物分别置于25 ℃孵育24小时或37 ℃孵育48小时后收集真菌细胞。
铁载体产生实验：为探究acuK与acuM在铁载体生物合成中的作用，我们采用铬天青S（Chrome Azurol S, CAS）固体与液体检测法，测定野生型、ΔacuK及ΔacuM菌株的铁载体产生量。首先将马尔尼菲篮状菌野生株与突变株接种至ANM液体培养基中培养7天。从培养上清液中检测胞外铁载体含量，从细胞裂解液中测定胞内铁载体含量。
巨噬细胞感染实验：采用人单核细胞系THP-1构建巨噬细胞感染模型，细胞于含10%胎牛血清（FBS, v/v）的RPMI 1640培养基（Life Technologies, 美国格兰德艾兰）中，于37 ℃、5% CO2条件下培养。将1×10^6个THP-1细胞接种至铺有无菌盖玻片的6孔板（用于吞噬实验）与12孔板（用于杀伤实验）中。用32 μM 佛波醇12-肉豆蔻酸酯13-乙酸酯（Phorbol 12-myristate 13-acetate, PMA）于37 ℃、5% CO2条件下诱导细胞分化24小时。随后将1×10^6个马尔尼菲篮状菌G809野生株（acuK+ acuM+）、ΔacuK及ΔacuM突变株的分生孢子加入巨噬细胞中，感染2小时。
对于吞噬实验，使用4%多聚甲醛固定巨噬细胞，并用1 mg/mL 荧光增白剂（calcofluor white）染色以观察真菌细胞壁。
针对接种后2小时的杀伤实验，收集巨噬细胞并用0.25% TritonX-100（Sigma-Aldrich）裂解。同时测定感染24小时后的巨噬细胞杀伤效果：感染2小时后，用PBS洗涤巨噬细胞三次，继续培养24小时后再裂解细胞。将回收的真菌细胞接种至合成葡萄糖琼脂培养基（SD：0.17% w/v 无氨基酸酵母氮源、2% w/v 葡萄糖、10 mM (NH4)2SO4、2% 琼脂）上，于37 ℃培养7–10天后统计菌落形成单位（CFU）。