Paracoccidioides brasiliensispresents metabolic reprogramming and secretes a serine proteinase during murine infection

Abstract

Paracoccidoides brasiliensis and Paracoccidioides lutzii, the etiologic agents of paracoccidioidomycosis, cause disease in healthy and immunocompromised persons in Latin America. We developed a method for harvesting P. brasiliensis yeast cells from infected murine lung to facilitate in vivo transcriptional and proteomic profiling. P. brasiliensis harvested at 6 h post-infection were analyzed using RNAseq and LC-MSE. In vivo yeast cells had 594 differentially expressed transcripts and 350 differentially expressed proteins. Integration of transcriptional and proteomic data indicated that early in infection (6 h), P. brasiliensis yeast cells underwent a shift in metabolism from glycolysis to β-oxidation, upregulated detoxifying enzymes to defend against oxidative stress, and repressed cell wall biosynthesis. Bioinformatics and functional analyses also demonstrated that a serine proteinase was upregulated and secreted in vivo. To our knowledge this is the first study depicting transcriptional and proteomic data of P. brasiliensis yeast cells upon 6 h post-infection of mouse lung.

Immunoproteomic Approach of Extracellular Antigens From Paracoccidioides Species Reveals Exclusive B-Cell Epitopes

“…These data provide the first large-scale identification of exoantigens of Paracoccidioides species. Some of these exoantigens have already been identified and described as antigens of pathogenic fungi, such as formamidase (Borges et al, 2005(Borges et al, , 2010, dihydrolipoyl dehydrogenase (Landgraf et al, 2017), HSP60 (Izacc et al, 2001;Cunha et al, 2002), HSP70 (Bromuro et al, 1998;Bisio et al, 2005), serine protease (Parente et al, 2010;Lacerda Pigosso et al, 2017), and isocitrate lyase (Cruz et al, 2011;Silva et al, 2019). This demonstrates the robustness of method used in our immunoproteomics analyses.…”

Metabolic Adaptation of Paracoccidioides brasiliensis in Response to in vitro Copper Deprivation

“…In interferon-gamma primate or non-macrophages, activation of the glyoxylate cycle, is a common strategy used by this fungus (Chaves et al, 2019). The gene induction o of the glyoxylate cycle and beta-oxidation is the strategy employed by P. brasiliensis during infection in mouse lung; this is also described during the infection of A. fumigatus conidia in neutrophils (Sugui et al, 2008;Lacerda Pigosso et al, 2017). Therefore, the activation of those alternative pathways for energy production could be a mechanism used for P. brasiliensis when in response to copper deprivation in vitro and in vivo.…”

“…Afterward, the cells were resuspended in an extraction buffer containing 20 mM Tris-HCl pH 8.8; 2 mM CaCl 2 with a mixture of nuclease and protease inhibitors (GE Healthcare). Following this step, the cells were distributed in tubes, glass beads were added, and the cells were disrupted on ice in a bead beater apparatus for 5 cycles of 30 s. Next, the cells were centrifuged at 10,000 g for 10 min at 4 • C, three times and the quantification of protein extracts was performed as described (Lacerda Pigosso et al, 2017).…”

“…Absorbance was measured at 412 nm using a plate reader. Triplicates were obtained for each condition, as previously described (Lacerda Pigosso et al, 2017). Results were considered statistically significant at p ≤ 0.01 by Student's t-test.…”

Metabolomics and Machine Learning Approaches Combined in Pursuit for More Accurate Paracoccidioidomycosis Diagnoses

“…During mouse lung infection, the yeast cells showed upregulation of enzymes involved in lipid oxidation. For example, acetyl coenzyme A (acetyl-CoA) and propionyl-CoA (both derived from lipid catabolism) are used during P. brasiliensis infection to fuel the glyoxylate cycle and provide a supply for synthesis of biomolecules (43). Besides, proteomic studies have also shown that yeast cells, once internalized by macrophages, present fatty acid degradation and its usage as fuel for survival inside phagocytic cells (44).…”