Iron Deprivation Modulates the Exoproteome in Paracoccidioides brasiliensis

Abstract

Fungi of the Paracoccidioides genus are the etiological agents of the systemic mycosis paracoccidioidomycosis and, when in the host, they find a challenging environment that is scarce in nutrients and micronutrients, such as Fe, which is indispensable for the survival of the pathogen. Previous studies have shown that fungi of this genus, in response to Fe deprivation, are able to synthesize and capture siderophores (Fe³⁺ chelators), use Fe-containing host proteins as a source of the metal, and use a non-canonical reductive pathway for Fe³⁺ assimilation. Despite all of these findings, there are still gaps that need to be filled in the pathogen response to metal deprivation. To contribute to the knowledge related to this subject, we obtained the exoproteome of Paracoccidioides brasiliensis (Pb18) undergoing Fe deprivation and by nanoUPLC-MS^E. One hundred forty-one proteins were identified, and out of these, 64 proteins were predicted to be secreted. We also identified the regulation of several virulence factors. Among the results, we highlight Cyb5 as a secreted molecule of Paracoccidioides in the exoproteome obtained during Fe deprivation. Cyb5 is described as necessary for the Fe deprivation response of Saccharomyces cerevisiae and Aspergillus fumigatus. Experimental data and molecular modeling indicated that Cyb5 can bind to Fe ions in vitro, suggesting that it can be relevant in the arsenal of molecules related to iron homeostasis in P. brasiliensis.

Keywords: Fe; cytochrome b5 (CYB5); microbial adaptation; nutritional immunity; secretome.

Figure 1

Summary of proteomic findings. P. brasiliensis yeast cells were subjected to Fe deprivation for 48 h, and fungal secreted proteins were identified by nanoUPCL-MS^E. A total of 141 proteins were identified. To increase the analysis stringency, the identified proteins were submitted to analysis of secretion prediction by online tools and data available in the literature, which resulted in the number of 64 proteins. Of those 64 proteins, 6 have potential ability to bind to iron.

Figure 2

Expression quantification of selected genes in Paracoccidioides brasiliensis by RT-qPCR. Quantitative RT-PCR data showing the transcript levels of cytochrome b5 (Cyb5) and gamma-glutamyl transpeptidase (Ggt2) in the presence and absence of iron at 6 and 24 h. The data were normalized using the gene encoding the 60S ribosomal protein L34 as the endogenous control and are presented as relative expression to the control. Data are expressed as the mean ± standard deviation of the triplicates of independent experiments. Student’s t-test was used for statistical comparisons. Error bars represent the standard deviation of three biological replicates, and * represents p ≤ 0.05.

Figure 3

Analyzing the expression dynamics of Paracoccidioides Cyb5 in iron deprivation. (A) Dot-blot analysis. Nitrocellulose membranes containing exoproteomes (FeSO₄ and BPS) were incubated with anti-Pb18Cyb5 (1:500) polyclonal antibodies or pre-immune sera (1:1,000). Pixel intensity was measured by densitometric analysis of immunoblotting dots using ImageJ software. Statistics analysis was performed through Student’s t-test. * Represents p ≤ 0.05.(B) Fluorescence microscopy of P. brasiliensis (Pb18) cells cultured in the presence or absence of iron for 24 h and subsequently incubated with primary antibody anti-Cyb5 and later with the secondary antibody anti-mouse IgG labeled with fluorescein isothiocyanate (FITC; Sigma). The data for fluorescence intensity evaluation were obtained through the AxioVision Software (Carl Zeiss). The values of fluorescence intensity (in pixels) and the standard error of each analysis were used to plot the graph. Data are expressed as mean ± standard error (represented using error bars); * represents p ≤ 0.05. One hundred cells of each condition were evaluated. All representative images in panel B were magnified ×400.

Figure 4

Cyb5 of Paracoccidioides brasiliensis is an Fe-binding protein. (A) Absorbance spectra of the purified recombinant Cyb5 protein in a lower oxidation state (green—sample previously treated with a reducing agent) and higher oxidation (purple). Controls: ferrous sulfate solution (dark blue), ferrous sulfate prepared in elution buffer (red), elution buffer (light blue), and purified recombinant Cb5 (orange). (B) The structural alignment of Cyb5 with iron-binding motifs resulted in two sites of iron binding for this protein with significant scores. Residues interacting with the iron ion are shown as sticks and labeled as His for histidine and Glu for glutamate. These are the amino acid residues less distant from the iron centers. Iron is shown as a brown sphere. (C) The structural alignment showed that His15 binds to iron in the distance of 2.1 Å, reaching a score above the threshold. The same residue may interact with iron more than once. Glutamic acid 11 (Glu11) and aspartic acid 20 (Asp20) bind to iron in the distance of 3.1 and 3.6 Å, respectively. (D) The structural alignment showed that the residues Glu56 and Glu59 bind to iron within the distance of 3.4 (A) Isoleucine 115 (Ile115) binds to iron with a lower score but still above the threshold. Bonds to the iron are shown as dotted yellow lines, with bond distances indicated next to the lines.