Interaction of the heterotrimeric G protein alpha subunit SSG-1 of Sporothrix schenckii with proteins related to stress response and fungal pathogenicity using a yeast two-hybrid assay

Abstract

BackgroundImportant biological processes require selective and orderly protein-protein interactions at every level of the signalling cascades. G proteins are a family of heterotrimeric GTPases that effect eukaryotic signal transduction through the coupling of cell surface receptors to cytoplasmic effector proteins. They have been associated with growth and pathogenicity in many fungi through gene knock-out studies. In Sporothrix schenckii, a pathogenic, dimorphic fungus, we previously identified a pertussis sensitive G alpha subunit, SSG-1. In this work we inquire into its interactions with other proteins.ResultsUsing the yeast two-hybrid technique, we identified protein-protein interactions between SSG-1 and other important cellular proteins. The interactions were corroborated using co-immuneprecipitation. Using these techniques we identified a Fe/Mn superoxide dismutase (SOD), a glyceraldehyde-3-P dehydrogenase (GAPDH) and two ion transport proteins, a siderophore-iron transporter belonging to the Major Facilitator Superfamily (MFS) and a divalent-cation transporter of the Nramp (natural resistance-associated macrophage protein) family as interacting with SSG-1. The cDNA's encoding these proteins were sequenced and bioinformatic macromolecular sequence analyses were used for the correct classification and functional assignment.ConclusionsThis study constitutes the first report of the interaction of a fungal G alpha inhibitory subunit with SOD, GAPDH, and two metal ion transporters. The identification of such important proteins as partners of a G alpha subunit in this fungus suggests possible mechanisms through which this G protein can affect pathogenicity and survival under conditions of environmental stress or inside the human host. The two ion transporters identified in this work are the first to be reported in S. schenckii and the first time they are identified as interacting with fungal G protein alpha subunits. The association of G protein alpha subunits to transport molecules reinforces the role of G proteins in the response to environmental signals and also highlights the involvement of fungal G protein alpha subunits in nutrient sensing in S. schenckii. These interactions suggest that these permeases could function as transceptors for G proteins in fungi.

Immunoproteomic analysis reveals a convergent humoral response signature in the Sporothrix schenckii complex

“…The higher the antigenic propensity scores, the more likely there is antigenic activity for the respective region. inside the human host [77]. 752 Moreover, a DIGE approach followed by MS/MS identifica-753 tion of differential whole-cell proteomes is currently being 754 used to compare S. schenckii, S. brasiliensis, S. globosa, and 755 S. mexicana in order to highlight aspects of the main dif-756 ferences in the biology and virulence of these species.…”

Proteins Potentially Involved in Immune Evasion Strategies in Sporothrix brasiliensis Elucidated by Ultra-High-Resolution Mass Spectrometry

“…CL, cellular localization; BP, biological process; MF, molecular function. The letters in the panels in the column at the right side of the figure represent references as follows: A, references 50 , 51 , 52 , and 53 ; B, reference 54 ; C, reference 55 ; D, references 59 , 60 , and 61 ; E, references 57 and 58 ; F, reference 24 ; G, references 28 , 29 , and 39 ; H, references 47 and 48 ; I, references 40 , 41 , 42 , and 43 .…”

“…We identified Mn superoxide dismutase in S. brasiliensis . The presence of SOD has been reported in S. schenckii ( 41 ), but the specific role of Mn SOD in virulence has yet to be addressed. In Cryptococcus gattii , virulence was reported to be dependent on Mn SOD, as mutants resulting from mutations of the Mn SOD gene presented attenuated virulence in vivo ( 42 ).…”

Identification of Metabolic Pathways Influenced by the G-Protein Coupled Receptors GprB and GprD in Aspergillus nidulans

“…A previous study reported the interaction of a G-alpha subunit of the fungal pathogen Sporothrix schenckii with two metal ion transporters, known as siderophores [43]. The permeases are generally expressed in response to stress conditions and iron deprivation.…”