Genetic and structural validation of phosphomannomutase as a cell wall target in Aspergillus fumigatus.

Wenxia Fang,Ling Lu,Yuanwei Zhang,Deborah E. A. Lockhart,Olawale G. Raimi,Daan M. F. van Aalten,Andrew T. Ferenbach

doi:10.1111/mmi.14706

Abstract

Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target.

Highlights

Aspergillus fumigatus is a ubiquitous and opportunistic filamentous fungus that causes life-threatening invasive aspergillosis with a high mortality rate in immunocompromised individuals such as bone marrow and solid organ transplant recipients (Brown et al, 2012; Kousha et al, 2011)
By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues
GDP-mannose biosynthesis is catalyzed by a cascade of three enzymes, starting from fructose 6-phosphate (Fru-6P), converted to mannose 6-phosphate (Man-6P) by mannose 6-phosphate isomerase (Pmi), isomerized to mannose 1-phosphate by phosphomannomutase (Pmm), and converted to GDP-mannose in the presence of GTP by GDP-mannose pyrophosphorylase (Gmp) (Sharma et al, 2014)

Summary

| INTRODUCTION

Aspergillus fumigatus is a ubiquitous and opportunistic filamentous fungus that causes life-threatening invasive aspergillosis with a high mortality rate in immunocompromised individuals such as bone marrow and solid organ transplant recipients (Brown et al, 2012; Kousha et al, 2011). AfPmmA requires glucose-1,6-bisphosphate (Glc-1,6-bisP, a phosphorylation activator) and Mg2+ for activity (Allen & Dunaway-Mariano, 2004; Qian et al, 2007) (Figure 1d) Taken together, these data suggest that A. fumigatus possesses a functional phosphomannomutase. Secreted protein catalyst in the transfer of the phosphoryl group to the Asp nucleophile (Figure S3) To detect whether these residues are required for AfPmmA catalytic activity, we performed site-directed mutagenesis to create two distinct point mutants (D25N and D27N) and assessed their kinetic properties. AfPmmA possesses potentially exploitable differences compared to the human orthologues

| DISCUSSION

D25 D231 D19 N218

Findings

| EXPERIMENTAL PROCEDURES