Journal: Virulence

Article Title: Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

doi: 10.1080/21505594.2024.2412750

Figure Lengend Snippet: Generation and verification of Hfl1p-tap (tandem affinity purification) construct. (a) Illustration outlining the integration of the Hfl1p-tap tag into the C. albicans genome. A heterozygous HFL1/hfl1∆ strain was generated using fusion PCR, employing the 5’ region of the HFL1 fragment, the 3’ region of the HFL1 fragment, and the LEU2 marker from the pSN40 plasmid. Confirmation of LEU2 prototrophic transformants (HFL-11) was achieved through PCR analysis, using the primers specified in Table S1. (b) Validation of Hfl1p-tap through Western blot analysis using an anti-TAP-Tag monoclonal antibody (1:5000). Whole-cell protein extracts from cultures grown in YPD (2% glucose) and YPG (2% glycerol) media were separated on a 10% SDS-PAGE gel. (c) Morphological resemblance of Hfl1p-tap strains (HTA6 and HTA9) to wild-type (WT) strain, evident through reduced pseudohyphae growth –the predominant growth form of the hfl1∆/hfl1∆ mutant – in YPD broth and agar medium. (d) Multiple sequence alignment of Hap5p and Hfl1p from C. albicans , and Dpb3p from S. cerevisiae (ScDpb3) was performed using EMBL-EBI’s clustal omega tool (version 1.2.4), highlighting the divergent sequence of Hfl1p from the other two proteins. Notably, the Hap4p-associated Domain#1 in Hap5p is absent in both Hfl1p and ScDpb3p.

Article Snippet: To optimize the antibody concentration for subsequent ChIP experiments, we evaluated various commercial products and found that anti-TAP-Tag mAb (ABclonal) at a 1:5000 dilution produced a clean product within the 38–52 kDa range, consistent with the 47 kDa size of TAP-tagged Hfl1p protein.

Techniques: Affinity Purification, Construct, Generated, Marker, Plasmid Preparation, Biomarker Discovery, Western Blot, SDS Page, Mutagenesis, Sequencing

Journal: Virulence

Article Title: Histone-like transcription factor Hfl1p in Candida albicans harmonizes nuclear and mitochondrial genomic network in regulation of energy metabolism and filamentation development

doi: 10.1080/21505594.2024.2412750

Figure Lengend Snippet: Integration of proteomic and phenotypic evidence highlighting Hfl1p’s role in carbon metabolism, mitochondrial respiration, and MDR transporters. (a) Proteomic data demonstrates reduced translation of mtDNA-encoded CI subunits (Nads), as well as nuclear-encoded CI subunits and CI regulator Goa1p, along with proteins associated with the TCA cycle in the HFL1 null mutant ( hfl1∆/hfl1∆ ). The downregulation of mitochondrial respiration and the TCA cycle is counterbalanced by elevated levels of proteins linked to non-glucose metabolism and mitochondrial transporters. (b) BN-page analysis reveals a significant decrease in the content of CI (complex I) in the hfl1∆/hfl1∆ mutant, particularly noticeable in YPD medium with 2% glucose when compared with WT and the dpb4∆/dpb∆ mutant. In YPG medium with 2% glycerol, the decrease in CI content is less evident. The quantifiable CI content from each strain, as determined by ImageJ analysis, is represented as relative ratios of CI/CIII and CI/CV intensities compared to WT [ , ]. The mean values obtained from three gel experiments underwent one-way ANOVA analysis. ”*” denotes statistical significance at p < 0.05. (c) The colocalization of Hfl1 (green) with mito-tracker red CMXRos (red). Tap-tagged Hfl1p is revealed by the primary antibody (mouse anti-TAP-Tag mAb) and secondary antibody (DyLight 488, goat anti-mouse IgG). Top row: HTA6, middle row: HTA9, bottom row: WT. Columns from left to right: DAPI (nuclei), mito-tracker red CMXRos, DyLight 488-Hfl1p, light, and merge. The orange arrows represent overlapping of Hfl1p with mitochondria, while the yellow arrows represent overlapping of Hfl1p with the nucleus.

Article Snippet: To optimize the antibody concentration for subsequent ChIP experiments, we evaluated various commercial products and found that anti-TAP-Tag mAb (ABclonal) at a 1:5000 dilution produced a clean product within the 38–52 kDa range, consistent with the 47 kDa size of TAP-tagged Hfl1p protein.

Techniques: Mutagenesis