Purpose: The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this study we sought to identify new regulatory components of oxidative stress/redox metabolism in pathogenic fungi associated primarily with virulence. Methods: In these studies bioinformatics was used to initially identify the tmpL gene in the Alternaria brassicicola and Aspergillus fumigatus genomes. Interpro analysis was used to predict conserved functional domains. Gene deletion experiments were carried out in both fungi in order to determine the role of TmpL in oxidative stress tolerance and virulence in plants and mammals. Co-localization experiments were performed in order to identify the location of TmpL protein in the fungal cell. Overexpression of the yeast yap1 ortholog in the tmpL deletion mutant background was performed in order to provide additional evidence that the virulence defects observed with tmpL deletion mutants were due to enhanced sensitivity to oxidative stress. Results: In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding/adenylation domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Data suggests that TmpL is a flavin-binding enzyme. Localization and gene expression analyses indicated that TmpL is associated with non-septal pore-associated Woronin bodies, specialized peroxisomes, and strongly expressed during conidiation and initial invasive growth. A. brassicicola and A. fumigatus tmpL deletion strains exhibited normal growth rates on a variety of media, but exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst, and hypersensitivity to oxidative stress when compared to wild-type/reconstituted strains. Moreover, A. brassicicola tmpL deletion strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on plants. Similarly, an A. fumigatus tmpL deletion strain was dramatically less virulent than wild-type and reconstituted strains in two different immune compromised murine models of invasive aspergillosis. Overexpression of the yap1 transcription factor complemented the majority of phenotypic defects observed. We are currently performing TmpL localization experiments, transcriptional profiling, and further characterizing the role of TmpL in virulence and modulation of the intracellular redox state in A. fumigatus. Conclusions: Collectively, we have discovered a protein with novel properties involved in virulence of plant and animal fungal pathogens. Our results 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.
Full conference title:
4th Advances Against Aspergillosis
- AAA 4th (2010)