Journal: mSphere

Article Title: MOB-mediated regulation of septation initiation network (SIN) signaling is required for echinocandin-induced hyperseptation in Aspergillus fumigatus

doi: 10.1128/msphere.00695-23

Figure Lengend Snippet: Deletion of the putative SIN regulators, sepM and mobA , alters colony morphology and early development. ( A ) Schematic of the proposed A. fumigatus SIN kinase cascade. The initiating kinase, SepH, receives activating signals from the fungus-specific GTPase, SpgA, which is regulated by the two-component GAP comprised of BubA and ByrA. SepH then activates SepL in a SepM-dependent manner which, in turn, activates SidB in a MobA-dependent manner. Schematic was made using Biorender. ( B ) Colony morphology of the control strain ( akuB-pyrG+ ), the sepM deletion mutant (∆ sepM ), the Δ sepM complemented strain (∆ sepM +sepM ), the mobA deletion mutant (∆ mobA ), and the Δ mobA complemented strain (∆ mobA +mobA ). Ten thousand conidia were spot-inoculated onto the center of glucose minimal media agar and allowed to grow for 4 days at 37°C. ( C ) Quantitation of colony diameter for each day post-inoculation. Colonies were grown as explained above for 4 days. Colony diameter was measured with calipers at the end of each 24-h period. Data presented are the average of three biological replicates per strain ± SD. Data were analyzed by two-way analysis of variance (ANOVA) with Turkey’s multiple comparison test. ( D ) Quantitative comparison of conidiation among all strains. Each strain was cultured as described above. On day 4, colony area (mm 2 ) was calculated for each culture and conidia were harvested in sterile distilled water. Conidia were counted via hemocytometer. Data are presented as total number of conidia normalized to total colony area ± SD. Data were analyzed by one-way ANOVA with Dunnett’s multiple comparison’s test. ( E ). Coverslip cultures were grown for each strain in triplicate for the indicated time. Each data point represents a different culture. After the indicated time point, coverslips were washed with phosphate buffered saline (PBS), fixed with a solution of 5% formalin, 2% triton-X by volume, treated with RNAse A, and stained with propidium iodide fluorescently stain nuclei. Between 60 and 100 germlings were evaluated for presence of one or more septa and number of nuclei. The average number of germlings with one or more septa and average number of nuclei per germling are shown. Data were analyzed by two-way ANOVA using Dunnett’s multiple comparisons test. ( F ) Coverslip cultures were grown for each strain in triplicate for the indicated time. Each data point represents a different culture. At each time point, coverslips were visualized by light microscopy and 200–400 conidia/germlings were evaluated for polarity establishment. The percentage of germlings with established polarity is shown. Data were analyzed by unpaired t -tests. All data in ( B–F ) represent three biological replicates. Error bars represent standard deviation. * = P < 0.05. ** = P < 0.01.

Article Snippet: P -values were calculated using a two-way ANOVA with Turkey’s test in GraphPad Prism 9.

Techniques: Control, Mutagenesis, Quantitation Assay, Comparison, Cell Culture, Sterility, Saline, Staining, Light Microscopy, Standard Deviation