TmpL, a Transmembrane Protein Required for Intracellular Redox Homeostasis and Virulence in a Plant and an Animal Fungal Pathogen

Abstract

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus ΔtmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola ΔtmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus ΔtmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola ΔtmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the ΔtmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola ΔtmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.

Author Summary

The critical roles of reactive oxygen species (ROS) in fungal development and virulence have been well established over the past half a century since the first experimental detection of hydrogen peroxide in fungal cells by Bach (1950). In the cell, ROS act as signaling molecules regulating physiological responses and developmental processes and are also involved in sophisticated virulence processes for many pathogenic fungi. Therefore, uncovering the biological roles of cellular ROS appears to be very important in understanding fungal development and virulence. Currently we have limited knowledge of how intracellular ROS are generated by fungal cells and which cellular ROS regulatory mechanisms are involved in establishing homeostasis. In this study we describe a novel protein, TmpL, involved in development and virulence in both plant and animal pathogenic fungi. In the absence of TmpL, dysregulation of oxidative stress homeostasis in both fungi caused developmental and virulence defects. Therefore, elucidating the role of TmpL presents an opportunity to uncover a common pathogenicity mechanism employed by both plant and animal pathogens and to develop efficient and novel therapeutics for both plant and animal fungal disease. Our findings provide new insights into mechanisms underlying the complex web of interactions between ROS and cell differentiation and the involvement of ROS for both plant and animal fungal pathogenesis.