Abstract
Aim:During infection development in the host, Paracoccidioides spp. faces the deprivation of micronutrients, a mechanism called nutritional immunity. This condition induces the remodeling of proteins present in different metabolic pathways. Therefore, we attempted to identify membrane proteins and their regulation by zinc in Paracoccidioides lutzii.Materials & methods:Membranes enriched fraction of yeast cells of P. lutzii were isolated, purified and identified by 2D LC–MS/MS detection and database search.Results & conclusion:Zinc deprivation suppressed the expression of membrane proteins such as glycoproteins, those involved in cell wall synthesis and those related to oxidative phosphorylation. This is the first study describing membrane proteins and the effect of zinc deficiency in their regulation in one member of the genus Paracoccidioides.
Highlights
Besides depicting the first large-scale depository of membrane proteins in the genus Paracoccidioides, this article highlights protein candidates potentially involved in the response to zinc limitation, a condition found in the host
The results presented here are from merged data of three replicates, leading to the identification of 746 proteins from the total membranes preparation of P. lutzii
We suggest that repression of those proteins upon zinc deprivation affects the glycosylation process in the endoplasmic reticulum in P. lutzii and, the profile of glycosylated proteins is less evident under zinc deprivation (Figure 4)
Summary
Yeast cells cultured in MMcM medium were centrifuged at 1200× g for 10 min at 4°C, frozen in liquid nitrogen and disrupted by maceration using a gral and pestle until a fine powder was obtained [31] After this step, the sample was transferred to a conical tube and resuspended in 50 mM Tris-HCl, pH 7.5. The data obtained after analysis by nanoUPLC-MSE and identification in ProteinLynx were submitted to in silico search in database in order to determine proteins subcellular location and association with cell membranes. (Sigma, catalog n.B8563) for 5 min under stirring and subsequently washed twice with PBS 1× [52] Both samples stained with ConA+FITC or aniline blue were visualized under a fluorescence microscope Fluorescence intensity was calculated as described above, for both dyes
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