Depicting the metabolism of Paracoccidioidesbrasiliensis during infection by transcriptional and proteomic approaches

KEYWORDS:

• fungal metabolism
• metabolic enzymes
• Paracoccidioides brasiliensis
• proteomic

This article refers to:

Paracoccidioides brasiliensis presents metabolic reprogramming and secretes a serine proteinase during murine infection

Species in the Paracoccidioides spp. complex are the causative agents of paracoccidioidomycosis (PCM), considered one of the most important systemic endemic mycosis in Latin America. The genus Paracoccidioides comprises 2 species, P. brasiliensis and P. lutzii with the former being classified into 4 different phylogenetic groups named S1, PS2, PS3 and PS4.^1-3 More recently, the S1 group was sub-divided into 2 lineages (S1a and S1b).⁴ The development of the disease depends on several factors dependent on both the host and the fungal virulence. Once the fungal pathogen enters in contact with the host, it should adapt itself to and override the various microenvironmental factors to survive inside the host. Fungal adaptation depends on its metabolism and capability to produce and secrete several molecules that play an important role in virulence. Among these molecules, Paracoccidioides species produce enzymes that participate in the most important pathways of central metabolism, involving carbohydrates, lipids, amino acids and nucleotides.⁵ In a previous study and using proteomic analysis, it was found that after internalization of P. brasiliensis into macrophages, a total of 308 differentially expressed proteins were detected in this fungal pathogen. The upregulated proteins included those related to β-oxidation of fatty acids and amino acids' catabolism, as well as proteins associated with the alternative carbon metabolism and enzymes involved in the gluconeogenesis pathway. 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. These interactions are involved in the adhesion and invasion process leading to Paracoccidioides infection.⁷

Paracoccidioides spp also has the capacity to defend itself against the oxidative and nitrosative stress molecules produced by the host´s immune cells especially those produced by phagocytic cells (mainly macrophages). The oxidative and nitrosative stress are characterized by production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), respectively. ROS comprises superoxide anion radical (O2-•), hydroxyl radical (•OH) and hydrogen peroxide (H₂O₂) while RNS comprises nitric oxide (NO•) and peroxynitrite (ONOO-), 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).^6,10-13 The important role in virulence of most of these detoxifying molecules has been demonstrated in elegant studies using knock-down or silenced P. brasiliensis strains obtained by antisense RNA (aRNA) approaches both in vitro and in vivo assays.^6,10-12 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.^6,10-12,14

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. These investigators used mass spectrometry-based approaches and mapped 440 phosphorylation sites in 230 P. brasiliensis proteins finding that phosphorylation at different sites determine the fungal response to oxidative stress.¹⁶

Several proteases are secreted by Paracoccidioides spp with some of them considered as virulence determinants that allow to the fungus to disseminate in its host. Among these molecules several have been described, namely, fructose 1,6-bisphosphate aldolase that binds to plasminogen and activates this into plasmin, which in turn, activates the fibrinolytic activity and also degrades ECM proteins, a fact that enables the fungus to adhere and invade host cells.¹⁷ A secreted aspartyl protease (PbSAP) has also been identified in P. brasiliensis.¹⁸ Aspartyl proteases constitute one of the 4 superfamilies of proteolytic enzymes, which have also been found in Candida albicans with at least 10 members identified, with SAPs 1–7 being differentially expressed during infection.^19,20 A serine-thiol protease with the capability to degrade laminin, fibronectin, type IV-collagen and proteoglycans, has also been identified in P. brasiliensis.^21,22

Of note, all the above reports using trasncriptomic or proteomic analysis have been performed by means of in silico or infected cell lines approaches. In this issue of Virulence, the authors of the article entitled “Paracoccidioides brasiliensis presents metabolic reprogramming and secretes a serine proteinase during murine infection” developed a method for harvesting P. brasiliensis yeasts from the lungs of infected mice to evaluate in vivo transcriptional and proteomic profiles.²³ A total of 594 differentially expressed transcripts and 350 differentially expressed proteins were annotated. As described before by the same group,⁶ in the investigation presented in this Virulence's issue, authors confirmed the upregulated expression of proteins related to metabolism including glycolysis, detoxifying enzymes and repressed cell wall biosynthesis in P. brasiliensis. Moreover, they also confirmed the upregulated expression of a serine protease, an enzyme involved in the invasion and dissemination of this fungal pathogen, shown to be secreted in vivo as described by the functional analysis already performed.^22,23 In sum, this article confirmed previous trasncriptomic and proteomic studies with the results presented here providing a better understanding of Paracoccidioides spp complex metabolism. Future studies using methodologies to silence or delete specific coding genes to obtain Paracoccidioides spp mutant strains, as well as in vivo functional studies would reveal key molecules that could be used as targets for developing new therapeutic strategies in PCM.

Disclosure of potential conflict of interest

No potential conflicts of interest were disclosed.