Browsing by Author "Puttikamonkul, Srisombat"

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In vivo hypoxia and a fungal alcohol dehydrogenase influence the pathogenesis of invasive pulmonary aspergillosis
(2011-07) Grahl, Nora; Puttikamonkul, Srisombat; Macdonald, Jeffrey M.; Gamcsik, Michael P.; Ngo, Lisa Y.; Hohl, Tobias M.; Cramer, Robert A.
Currently, our knowledge of how pathogenic fungi grow in mammalian host environments is limited. Using a chemotherapeutic murine model of invasive pulmonary aspergillosis (IPA) and 1H-NMR metabolomics, we detected ethanol in the lungs of mice infected with Aspergillus fumigatus. This result suggests that A. fumigatus is exposed to oxygen depleted microenvironments during infection. To test this hypothesis, we utilized a chemical hypoxia detection agent, pimonidazole hydrochloride, in three immunologically distinct murine models of IPA (chemotherapeutic, X-CGD, and corticosteroid). In all three IPA murine models, hypoxia was observed during the course of infection. We next tested the hypothesis that production of ethanol in vivo by the fungus is involved in hypoxia adaptation and fungal pathogenesis. Ethanol deficient A. fumigatus strains showed no growth defects in hypoxia and were able to cause wild type levels of mortality in all 3 murine models. However, lung immunohistopathology and flow cytometry analyses revealed an increase in the inflammatory response in mice infected with an alcohol dehydrogenase null mutant strain that corresponded with a reduction in fungal burden. Consequently, in this study we present the first in vivo observations that hypoxic microenvironments occur during a pulmonary invasive fungal infection and observe that a fungal alcohol dehydrogenase influences fungal pathogenesis in the lung. Thus, environmental conditions encountered by invading pathogenic fungi may result in substantial fungal metabolism changes that influence subsequent host immune responses.
TmpL, a transmembrane protein required for intracellular redox homeostasis and virulence in a plant and an animal fungal pathogen
(2009-11) Kim, Kwang-Hyung; Willger, Sven D.; Park, Sang-Wook; Puttikamonkul, Srisombat; Grahl, Nora; Cho, Yangrae; Mukhopadhyay, Biswarup; Cramer, Robert A.; Lawrence, Christopher B.
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 DtmpL 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 DtmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus DtmpL 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 DtmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the DtmpL 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 DtmpL 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.
Trehalose-6-phosphate is required for metabolism and virulence in the human fungal pathogen Aspergillus fumigatus
(Montana State University - Bozeman, College of Agriculture, 2012) Puttikamonkul, Srisombat; Chairperson, Graduate Committee: Robert Cramer; Sven D. Willger, Nora Grahl, Navid Movahed, Brian Bothner, Steven Park, Padmaja Paderu, David S. Perlin, John R. Perfect, and Robert A. Cramer Jr. were co-authors of the article, 'Trehalose 6-phosphate phosphatase is required for cell wall integrity and fungal virulence, but not trehalose biosynthesis in the human fungal pathogen Aspergillus fumigatus' in the journal 'Molecular microbiology' which is contained within this thesis.; Kelly Shepardson, Sven D. Willger, Nora Grahl, Jonathan Hilmer, Brian Bothner, Vishu Kumar Amanianda, Jean-Paul Latge, John R. Perfect, and Robert A. Cramer Jr. were co-authors of the article, 'The trehalose pathway intermediate, T6P, regulates cell wall composition and virulence in Aspergillus fumigatus' in the journal 'Blood' which is contained within this thesis.
High mortality rates associated with Invasive Pulmonary Aspergillosis (IPA), commonly caused by the mold Aspergillus fumigatus, have dramatically increased in immunocompromised patients during the past 30 years. With limited antifungal drugs available and inconsistent outcomes associated with current antifungal drug treatment, much effort is being focused on new antifungal drug development. A. fumigatus has evolved multifactorial mechanisms to survive various stress conditions encountered in environment and in vivo during infection. Targeting the biochemical pathways utilized by the fungus to adapt to stress conditions is one proposed approach for development of new antifungal drugs. Biosynthesis of the disaccharide trehalose is one such target that is not found in mammals. This dissertation aimed to characterize the role of the trehalose biosynthesis pathway in the biology and virulence of A. fumigatus. My objectives were to identify the function of putative enzyme encoding genes involved in the TPS1/TPS2 pathway and establish the contribution of each protein to the virulence of A. fumigatus in clinically relevant IPA murine models. My data suggests that the Trehalose-6-phosphate intermediate of the TPS1/TPS2 pathway plays a critical role in regulating fungal metabolic homeostasis and integrity of fungal cell wall. A mutant deficient in the TPS2 ortholog, OrlA, displayed increased sensitivity to cell wall perturbing drugs and importantly was attenuated in virulence in two murine models of IPA. My data further suggests that the attenuated virulence phenotype is directly linked to these changes in the fungal cell wall that alter the innate immune response to this fungal strain. In contrast, trehalose itself while having a general role in stress protection does not have a role in virulence in IPA models. Finally, my data suggest intricate links between the mobilization of trehalose and accumulation of T6P that also affect fungal metabolism and cell wall homeostasis via the activity of trehalose phosphorylase enzymes. In conclusion, my data supports the hypothesis that the trehalose biosynthesis pathway is a potential target for antifungal drug development in A. fumigatus particularly at the level of TPS2 activity. However, the underlying host immune status must also be taken into account when targeting this key fungal metabolic pathway.