Dephosphorylation of the Core Septin, AspB, in a Protein Phosphatase 2A-Dependent Manner Impacts Its Localization and Function in the Fungal Pathogen Aspergillus fumigatus.

José M Vargas-Muñiz,William J Steinbach,Yohannes Asfaw,Martin A Moseley,Praveen R Juvvadi,Hilary Renshaw,Greg Waitt,Amber D Richards,Erik J Soderblom

doi:10.3389/fmicb.2016.00997

Abstract

Septins are a conserved family of GTPases that form hetero–oligomeric complexes and perform diverse functions in higher eukaryotes, excluding plants. Our previous studies in the human fungal pathogen Aspergillus fumigatus revealed that the core septin, AspB, a CDC3 ortholog, is required for septation, conidiation, and conidial cell wall organization. Although AspB is important for these cellular functions, nothing is known about the role of kinases or phosphatases in the posttranslational regulation and localization of septins in A. fumigatus. In this study, we assessed the function of the Gin4 and Cla4 kinases and the PP2A regulatory subunit ParA, in the regulation of AspB using genetic and phosphoproteomic approaches. Gene deletion analyses revealed that Cla4 and ParA are indispensable for hyphal extension, and Gin4, Cla4, and ParA are each required for conidiation and normal septation. While deletion of gin4 resulted in larger interseptal distances and hypervirulence, a phenotype mimicking aspB deletion, deletion of cla4 and parA caused hyperseptation without impacting virulence, indicating divergent roles in regulating septation. Phosphoproteomic analyses revealed that AspB is phosphorylated at five residues in the GTPase domain (S134, S137, S247, T297, and T301) and two residues at its C-terminus (S416 and S461) in the wild-type, Δgin4 and Δcla4 strains. However, concomitant with the differential localization pattern of AspB and hyperseptation in the ΔparA strain, AspB remained phosphorylated at two additional residues, T68 in the N-terminal polybasic region and S447 in the coiled-coil domain. Generation of nonphosphorylatable and phosphomimetic strains surrounding each differentially phosphorylated residue revealed that only AspBmt-T68E showed increased interseptal distances, suggesting that dephosphorylation of T68 is important for proper septation. This study highlights the importance of septin phosphorylation/dephosphorylation in the regulation of A. fumigatus hyphal septation.

Highlights

Septins, a conserved family of GTPases, are involved in a variety of critical cellular functions, ranging from cell division to cell wall maintenance (Momany et al, 2001; Alvarez-Tabares and Perez-Martin, 2010; Kozubowski and Heitman, 2010; Lindsey et al, 2010; Hernandez-Rodriguez et al, 2012, 2014)
Our previous studies showed that A. fumigatus septins play a pleiotropic role in septation, conidiation, and response to anticell wall agents (Vargas-Muniz et al, 2015)
To test whether the kinases and the phosphatase 2A (PP2A) subunit deletion strains phenocopy this increase in susceptibility, the respective deletion strains were cultured in the presence of the cell wall stressor Congo Red as well as the β-glucan synthase inhibitor, caspofungin, and the chitin synthase inhibitor, nikkomycin Z

Summary

Introduction

A conserved family of GTPases, are involved in a variety of critical cellular functions, ranging from cell division to cell wall maintenance (Momany et al, 2001; Alvarez-Tabares and Perez-Martin, 2010; Kozubowski and Heitman, 2010; Lindsey et al, 2010; Hernandez-Rodriguez et al, 2012, 2014). In the pathogenic fungus Candida albicans there are seven septin genes, all of which have orthologs in the model yeast Saccharomyces cerevisiae, while Aspergillus fumigatus has only five septin genes (Warenda and Konopka, 2002; Pan et al, 2007; Juvvadi et al, 2011b). A. fumigatus aspA, aspB, aspC, and aspD are orthologous to S. cerevisiae CDC11, CDC3, CDC12, and CDC10, respectively; while aspE is absent in S. cerevisiae (Pan et al, 2007; Lindsey et al, 2010; Juvvadi et al, 2011b). Deletion analyses in A. fumigatus revealed that none of the septins are required for growth under basal conditions, yet septins AspA, AspB, AspC, and AspE play a key role in regular septation and AspA, AspB, AspC, and AspD regulate conidiation (Vargas-Muniz et al, 2015). Deletion of aspB resulted in hypervirulence in an invertebrate model of invasive aspergillosis, as well as increased susceptibility and AspB mislocalization following exposure to anti-cell wall agents

Methods

Results

Conclusion