Macrophage Interaction with Paracoccidioides brasiliensis Yeast Cells Modulates Fungal Metabolism and Generates a Response to Oxidative Stress

Abstract

Macrophages are key players during Paracoccidioides brasiliensis infection. However, the relative contribution of the fungal response to counteracting macrophage activity remains poorly understood. In this work, we evaluated the P. brasiliensis proteomic response to macrophage internalization. A total of 308 differentially expressed proteins were detected in P. brasiliensis during infection. The positively regulated proteins included those involved in alternative carbon metabolism, such as enzymes involved in gluconeogenesis, beta-oxidation of fatty acids and amino acids catabolism. The down-regulated proteins during P. brasiliensis internalization in macrophages included those related to glycolysis and protein synthesis. Proteins involved in the oxidative stress response in P. brasiliensis yeast cells were also up-regulated during macrophage infection, including superoxide dismutases (SOD), thioredoxins (THX) and cytochrome c peroxidase (CCP). Antisense knockdown mutants evaluated the importance of CCP during macrophage infection. The results suggested that CCP is involved in a complex system of protection against oxidative stress and that gene silencing of this component of the antioxidant system diminished the survival of P. brasiliensis in macrophages and in a murine model of infection.

Depicting the metabolism of Paracoccidioides brasiliensis during infection by transcriptional and proteomic approaches

“…As it concerns the downregulated proteins included those related to glycolysis and protein synthesis. 6 Interestingly, it has been described that most of the Paracoccidioides spp enzymes that participate in both the glycolytic pathway and the tricarboxylic acid (TCA) cycle including glyceraldehyde-3-phosphate dehydrogenase (GADPH), enolase (ENO), fructose-1-6-bisphosphate aldolase (FBA), triose phosphate isomerase (TPI), malate synthase (MLS) and isocitrate lyase (ICL), function as moonlight proteins, this is, they are multifunctional proteins capable of accomplishing different functions often unrelated. 7 Thus, these glycolytic enzymes allow Paracoccidioides spp binding to extracellular matrix (ECM) proteins such as laminin, fibronectin, fibrinogen, type I and IV collagens and plasminogen, among others.…”

“…8 These oxidative and nitrosative molecules act on fungal cells through the inactivation of proteins, lipids and cellular enzymes inhibiting respiration, and damaging the DNA and membranes thus leading to the fungal death. 6,9 Therefore, several reports have been shown that Paracoccidioides spp has a powerful antioxidant defense system that allows this fungal pathogen to survive ROS and RNS production; this system includes expression/production of several enzymes such as catalases (CATs), superoxide dismutases (SODs), alternative oxidase (AOX), cytochrome c peroxidase (CCP) and thioredoxins (THX).…”

“…6,[10][11][12] Moreover, Parente-Rocha et al using proteomic analysis after internalization of P. brasiliensis into macrophages found upregulation of proteins involved in the oxidative stress response such as SODs, THX and CCP. 6 Of note, all P. brasiliensis muted strains obtained through the use of aRNA methodology for SODs, AOX, CATs and CCP, showed diminished survival into macrophages as well as in experimental mouse models.…”

“…6,[10][11][12] Moreover, Parente-Rocha et al using proteomic analysis after internalization of P. brasiliensis into macrophages found upregulation of proteins involved in the oxidative stress response such as SODs, THX and CCP. 6 Of note, all P. brasiliensis muted strains obtained through the use of aRNA methodology for SODs, AOX, CATs and CCP, showed diminished survival into macrophages as well as in experimental mouse models. 6,[10][11][12]14 Most of the vital processes undergone by the eukaryotic cells such as cell cycle, survival, adhesion and proliferation, among other processes, are governed by the signals transduction that in turn are addressed by protein phosphorylation at post-translational levels.…”

“…6 Of note, all P. brasiliensis muted strains obtained through the use of aRNA methodology for SODs, AOX, CATs and CCP, showed diminished survival into macrophages as well as in experimental mouse models. 6,[10][11][12]14 Most of the vital processes undergone by the eukaryotic cells such as cell cycle, survival, adhesion and proliferation, among other processes, are governed by the signals transduction that in turn are addressed by protein phosphorylation at post-translational levels. 15 Along these lines, Chaves et al investigated the influence of phosphorylative events during the P. brasiliensis adaptation to oxidative stress.…”