Loss of Septation Initiation Network (SIN) kinases blocks tissue invasion and unlocks echinocandin cidal activity against Aspergillus fumigatus

PLoS Pathog. 2021 Aug 9;17(8):e1009806. doi: 10.1371/journal.ppat.1009806. eCollection 2021 Aug.

Abstract

Although considered effective treatment for many yeast fungi, the therapeutic efficacy of the echinocandin class of antifungals for invasive aspergillosis (IA) is limited. Recent studies suggest intense kinase- and phosphatase-mediated echinocandin adaptation in A. fumigatus. To identify A. fumigatus protein kinases required for survival under echinocandin stress, we employed CRISPR/Cas9-mediated gene targeting to generate a protein kinase disruption mutant library in a wild type genetic background. Cell wall and echinocandin stress screening of the 118 disruption mutants comprising the library identified only five protein kinase disruption mutants displaying greater than 4-fold decreased echinocandin minimum effective concentrations (MEC) compared to the parental strain. Two of these mutated genes, the previously uncharacterized A. fumigatus sepL and sidB genes, were predicted to encode protein kinases functioning as core components of the Septation Initiation Network (SIN), a tripartite kinase cascade that is necessary for septation in fungi. As the A. fumigatus SIN is completely uncharacterized, we sought to explore these network components as effectors of echinocandin stress survival. Our data show that mutation of any single SIN kinase gene caused complete loss of hyphal septation and increased susceptibility to cell wall stress, as well as widespread hyphal damage and loss of viability in response to echinocandin stress. Strikingly, mutation of each SIN kinase gene also resulted in a profound loss of virulence characterized by lack of tissue invasive growth. Through the deletion of multiple novel regulators of hyphal septation, we show that the non-invasive growth phenotype is not SIN-kinase dependent, but likely due to hyphal septation deficiency. Finally, we also find that echinocandin therapy is highly effective at eliminating residual tissue burden in mice infected with an aseptate strain of A. fumigatus. Together, our findings suggest that inhibitors of septation could enhance echinocandin-mediated killing while simultaneously limiting the invasive potential of A. fumigatus hyphae.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antifungal Agents / pharmacology
  • Aspergillosis / drug therapy*
  • Aspergillosis / enzymology
  • Aspergillosis / microbiology
  • Aspergillosis / pathology
  • Aspergillus fumigatus / drug effects*
  • Aspergillus fumigatus / enzymology
  • Echinocandins / pharmacology*
  • Female
  • Fungal Proteins / metabolism*
  • Lung / drug effects*
  • Lung / microbiology
  • Lung / pathology
  • Mice
  • Protein Kinases / deficiency*

Substances

  • Antifungal Agents
  • Echinocandins
  • Fungal Proteins
  • Protein Kinases