X Guruceaga1*, A Martin-Vicente1, ACO Souza2, AV Nywening 1, H Thorn1, J Xie1, W Ge1, BM Peters1,3, JR Fortwendel1,3
1Department of Clinical Pharmacy and Translational Science, University of Tennessee Health Science Center, Memphis, USA
2Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children’s Research Hospital, Memphis, USA
3Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, USA
Full conference title:
10th Advances Against Aspergillosis and Mucormycosis
Date: 2 February 2022
Aspergillus fumigatus is the main causative agent of invasive aspergillosis. The therapeutic armamentarium to fight against this disease is limited. Therefore, we sought to identify genetic pathways that might serve as novel therapeutic targets through generating and studying an A. fumigatus protein kinase disruption mutant library. The aim of the current work is to understand the importance of the Septation Initiation Network (SIN) kinase genes, sepH, sepL and sidB,in A. fumigatus pathobiology.
Cell wall characterization was performed using standard Aspergillus culture techniques. Staining of exposed chitin was performed using Wheat Germ Aggluntinin-FITC and β-glucan detected using an Fc-hDectin-1a recombinant molecule. Germination, septation and mitosis were analyzed by microscopy using calcofluor white and propidium iodide staining. RNAseq analyses were performed using mature hyphae grown in minimal media for 24 hours. Total RNA was extracted using TRIzol coupled with a RNeasy Qiagen kit, following manufacturer’s instructions.
We previously found the SIN kinase genes, sepH, sepL and sidB, to be essential for hyphal septation and for survival under cell wall stress. Our previous work also revealed that loss of hyphal septation resulted in almost complete avirulence characterized by lack of tissue invasion. Interestingly, co-culture of mutant and wild type conidia with a macrophage cell line revealed reduced ability of the SIN kinase mutants to elicit pro-inflammatory signaling evidenced by reduced IL-1β and TNFα release. To see if the cell wall hypersusceptibility and reduced immunogenicity phenotypes of the SIN kinase mutants were due to altered cell wall formation or hyphal development, we performed in vitro phenotypic characterization of the mutant strains coupled with RNAseq profiling. Interestingly, conidia of the mutant strains were found to initiate germination earlier than wild type and hyphae formed by the mutant germlings were significantly longer than the wild type at the same developmental timepoint. In contrast, the mitotic rate was similar between all the strains, suggesting that loss of SIN activity uncoupled the processes of mitosis polarized morphogenesis. Hyphal staining revealed altered distribution of chitin cell wall deposition and reduced β-glucan levels in the Sin kinase mutants. RNAseq analyses identified 997, 625 and 337 genes from the ΔsepH, sepL-1, and sidB-1 mutant strains, respectively, that
were differentially expressed when compared to the wild type. Of these genes, only 28 were downregulated in common between the three mutant strains. Gene Ontology enrichment analyses revealed that 45% of these genes were related with transport processes, 34% with RNA metabolic process and 28% with ribosome biogenesis.
Our results suggest that SIN kinase activity is essential for normal germination and mitosis and for proper cell wall formation. Although no direct transcriptional link to cell wall biosynthesis was uncovered, RNAseq analyses suggest hyphal septation is linked to cellular transport, RNA metabolism and ribosome biogenesis.
Abstract Number: 47
Conference Year: 2022
Link to conference website: https://aaam2022.org/
URL Conference abstract: