A novel GAL4-like transcriptional regulator modulate the azoles sensitivity of Exophiala dermatitidis

Abstract Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Exophiala dermatitidis is an emerging opportunistic pathogen among the ever-increasing numbers of immunocompromised hosts. Understanding its transmission mechanism, pathogenicity, and potential azole-resistant mechanism is important for better understanding of this fatal pathogen and benefits future diagnosis, and treatment. By using a PCR-based fragment fusion approach, we applied phosphotransferase (HPH) resistance gene which amplified from pAN7-1 as a selective gene for generating replacement cassettes. Combined with electroporation transformation and screening on selection medium containing 50 μg/ml HMB, we obtained the gene knockout strain Δ GAL4, a novel GAL4-like transcriptional factor (XP_009154426.1) encoding gene (GeneID:20 306 783) accurately deleted. Methods Growth curve determination: Take 200 μL of 2 × 10⁴ CFU/ml bacterial suspension and 200 μL liquid sabourand medium in EP tubes. Shaking at 200 rpm 37°C for 0 h, 1 h, 2 h, 3 h, 4 h, 8 h, 12 h, 16 h, 18 h, 24 h, 30 h, 36 h, 48 h. Then add 200 μL cultured bacterial suspension to a 96-well plate and repeat three times. Meanwhile set up blank medium control wells. By using an enzyme marker to measure the absorbance at 504 nm, analyzing with SPSS, and then graphing with GraphPad software. Morphological observations: Activated strains on Sabourand microcultures and incubated at 37 °C for 3d. Observe morphology by using fluorescent microscopy to compare the different strains of wild and knockout strains, and using lactic acid cotton phenol blue/fluorescent dye filming observe the morphology of conidia. E-test drug sensitivity method: Spread fresh bacterial suspension of ΔGAL4 and ATCC34100 containing 1 × 10⁶CFU/ml spores on RPMI 1640 agar plates with sterile cotton swabs, and place drug sensitivity test strips [itraconazole (Itraconazole Liofilchem, ICZ 92148), posaconazole (Posaconazole Liofilchem (PCZ 92152), voriconazole (Mérieux, VRC 532800)] in the center plate, after sealing with a parafilm, incubating in 37°C for 36-48 h, and read the values at the tangent of the drug susceptibility strip and the antibacterial circle. Results The results revealed that the ΔGAL4 showed almost no difference in growth and morphology compared with wild strain, but its sensitivity to azoles was altered. We applied E-test to evaluate the MIC changes of azoles. The MIC of itraconazole, voriconazole, and posaconazole against ΔGAL4 was 0.5 μg/ml, 0.125 μg/ml, and 0.25 μg/ml; against ATCC34100 was 2 μg/ml, 0.19 μg/ml, and 0.38 μg/ml, respectively. △GAL4 MIC suggests that ∆GAL4 showed more sensitivity to azoles compared to its wild-type strain. Conclusion The present results suggested that a novel GAL4-like transcriptional factor may play an important role in azole sensitivity of E. dermatitidis, which may be through modulating efflux pumps or Cyp51A. Further investigation of this GAL4 related modulating pathway is needed.