The Aspergillus fumigatus morphogenetic NDR kinase, CotA, partially exerts carbon preference through the translational repressor, ssdA

Author:

Adela Martin-Vicente (US)

Abstract:

Background: Aspergillus fumigatus SsdA is a putative translational repressor regulating growth, cell wall integrity, and virulence. Orthologs of SsdA are known to be negatively regulated by the conserved Nuclear Dbf2-Related (NDR) kinases Cbk1 and COT-1 in Saccharomyces cerevisiae and Neurospora crassa, respectively. We previously identified the A. fumigatus Cbk1 ortholog, CotA, as an important virulence component in mouse models of invasive aspergillosis. Although the underlying virulence mechanism is unknown, we found CotA to regulate invasive hyphal growth in response to host-relevant carbon sources. Strains carrying cotA gene disruptions fail to grow on non-sugar carbon sources that are readily available in the host lung environment, such as acetate and amino acids. The main objective of this work was to determine if CotA orchestrates growth in non-preferred carbon sources through the conserved downstream effector, SsdA.

Methods: For growth rate analyses, 104 conidia were inoculated in the center of agar plates and colony diameters measured every 24 hours for 4 days. For biomass quantification, 50 ml of minimal media containing 1% glucose, 1% potassium acetate, 1% ethanol or 1% casaminoacids, as the sole carbon sources, were inoculated with 2×107 conidia and incubated at 37 °C for 24 hours in static conditions. Mycelia were recovered and lyophilized weights recorded. Deletion of ssdA was accomplished by replacing the predicted ssdA ORF with a phleomycin resistance cassette in the wild type CEA10 and cotA disruption (cotA-1) backgrounds, using CRISPR-Cas9. Western blot analyses were performed using a custom antibody raised against the A. fumigatus CotA protein.

Results: Loss of ssdA in CEA10 (ΔssdA) caused significant growth defects in all conditions tested, reducing colony diameter of mature cultures by 30-50% depending on the carbon source. In contrast, deletion of ssdA in the cotA-1 disruption mutant (ΔssdA/cotA-1) caused only a minor growth reduction in the presence of glucose. Strikingly, when compared to the parental cotA-1 disruption mutant, this double mutation resulted in significant growth recovery in non-sugar carbon sources. These results correlated with biomass accumulation observations, where the ΔssdA/cotA-1 mutant displayed a significant increase in biomass in comparison to the cotA-1 parental strain under similar conditions. Therefore, loss of ssdA partially restored growth to the cotA-1 mutant in alternative carbon sources. As it is a putative translational repressor, we next sought to test if SsdA might function in a feedback loop to regulate CotA protein abundance in response to carbon source. In the wild type, we observed that culture in acetate resulted in only mild reductions in CotA protein abundance when compared to glucose culture conditions. Loss of ssdA did not impact CotA abundance in glucose, when compared to the wild type. In contrast, we observed a significant reduction of CotA abundance in ΔssdA under acetate versus glucose culture.

Conclusions: Together, our findings support the hypothesis that the conserved translational repressor, SsdA, operates downstream of CotA to partially regulate the carbon source-dependent roles of CotA signaling in A. fumigatus. However, this regulatory mechanism is unlikely to be through direct feedback regulation of carbon source-responsive CotA translation.

Abstract Number: 46

Conference Year: 2024

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