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.

NosA, a transcription factor important in Aspergillus fumigatus stress and developmental response, rescues the germination defect of a laeA deletion

“…We were curious to test the virulence of the nosA mutants as characteristics of both the overexpression (increased resistance to menadione) and deletion (increased vegetative growth and germination rates) nosA strains have been correlated with virulence in other mutants (Ejzykowicz et al, 2009; Kim et al, 2009; reviewed in Abad et al, 2010). Because the ΔlaeA mutant (with increased nosA expression) is hypovirulent (Bok et al, 2005, Sugui et al 2007, Ben-Ami et al 2009), we predicted that the ΔnosA strain was more likely to show increased virulence than the OE::nosA strain.…”

VeA Regulates Conidiation, Gliotoxin Production, and Protease Activity in the Opportunistic Human Pathogen Aspergillus fumigatus

“…A 1.2-kb 3=-UTR fragment was also amplified from genomic DNA with primers 3=F-Afu579 and 3=R-Afu580 ( ) was amplified from plasmid p1439 (84) using primers Afu5PyrG581 and Afu3PyrG582 (Table 2). pyrG was utilized as transformation marker, achieving a complete gene replacement of veA in CEA17, a commonly used pyrG auxotroph derived from the wild-type pathogenic isolate CBS144-89 (32,40,80).…”

Phosphoribosylamidotransferase, the first enzyme for purine de novo synthesis, is required for conidiation in the sclerotial mycoparasite Coniothyrium minitans

“…There are several studies reporting that the coenzymes such as FDA or NADH are very important in conidiation and other developmental processes (Stuart and Stephanie, 1973;Soid-Raggi et al, 2006;Kim et al, 2009). Thus, we propose a hypothesis that the shortage of purines may cause a series of shortages of adenosinerelated important components, e.g.…”