Involvement of an Alternative Oxidase in Oxidative Stress and Mycelium-to-Yeast Differentiation in Paracoccidioides brasiliensis

Abstract

Paracoccidioides brasiliensis is a thermodimorphic human pathogenic fungus that causes paracoccidioidomycosis (PCM), which is the most prevalent systemic mycosis in Latin America. Differentiation from the mycelial to the yeast form (M-to-Y) is an essential step for the establishment of PCM. We evaluated the involvement of mitochondria and intracellular oxidative stress in M-to-Y differentiation. M-to-Y transition was delayed by the inhibition of mitochondrial complexes III and IV or alternative oxidase (AOX) and was blocked by the association of AOX with complex III or IV inhibitors. The expression of P. brasiliensis aox (Pbaox) was developmentally regulated through M-to-Y differentiation, wherein the highest levels were achieved in the first 24 h and during the yeast exponential growth phase; Pbaox was upregulated by oxidative stress. Pbaox was cloned, and its heterologous expression conferred cyanide-resistant respiration in Saccharomyces cerevisiae and Escherichia coli and reduced oxidative stress in S. cerevisiae cells. These results reinforce the role of PbAOX in intracellular redox balancing and demonstrate its involvement, as well as that of other components of the mitochondrial respiratory chain complexes, in the early stages of the M-to-Y differentiation of P. brasiliensis.Paracoccidioides brasiliensis, which is a thermally dimorphic fungus, is the etiological agent of paracoccidioidomycosis (PCM), which is the most prevalent human systemic mycosis in Latin America (5, 52) and affects almost 10 million individuals in Latin America (54). It is acquired by the inhalation of airborne microconidia, which reach the pulmonary alveolar epithelium and transform into the pathogenic yeast form (52,54,59). The human form of PCM that is caused by this fungus is characterized by a range of clinical manifestations, ranging from asymptomatic forms to severe, disseminated, and often fatal disease. Usually, fibrosis sequelae in the affected organs may interfere with the well-being of the patient (50).During infection, thermodimorphic fungi, such as Histoplasma capsulatum, Blastomyces dermatitidis, and P. brasiliensis, differentiate from a mycelial into a pathogenic yeast form, a transition that can also be induced (in vitro) by temperature changes from between 23 and 26°C to between 35 and 37°C. In addition, these pathogens are often subjected to significant environmental stresses, including exposure to the reactive oxygen species (ROS) and reactive nitrogen species (RNS) that are produced by the host cells (6,27,45).Elevated sublethal temperatures (thermal stress or heat shock) increase ROS generation and oxidative damage in a variety of eukaryotic cells (16,49,60,73). Mitochondria are the primary intracellular sources of ROS, which are generated in respiratory chain complexes I and III and can damage biomolecules, such as nucleic acids, lipids, and proteins (20)(21)(22). In order to control ROS levels, cells employ diverse detoxification mechanisms, including superoxide dismutase, catalase, the glutathione/thioredoxin...

Mitochondria and Fungal Pathogenesis: Drug Tolerance, Virulence, and Potential for Antifungal Therapy

“…Whether this discrepancy is due to differences in strain backgrounds of C. albicans or the nature of the mitochondrial mutations remains to be determined. In the dimorphic pathogen P. brasiliensis, inhibition of mitochondrial respiration blocks the switch from filamentous to yeast morphology that is important for virulence (56). In conclusion, the available data from both direct and indirect studies support the notion that wild-type mitochondrial function is required for virulence.…”

“…Indirect evidence for a role of mitochondria in the survival of fungal pathogens within the host includes a requirement for mitochondrial function for metabolic pathways necessary for virulence (such as the glyoxylate cycle and gluconeogenesis) (2,72), the activation of genes required for mitochondrial respiration during colonization of the central nervous system by C. neoformans (80), and the involvement of mitochondria in sur-vival under oxidative stress in C. albicans, C. neoformans, A. fumigatus, and Paracoccidioides brasiliensis (1,54,56,62), as well as the role of mitochondria in fungal morphogenesis (1,46,56,57,87). With respect to morphogenesis, C. albicans mutants lacking the mitochondrial protein Goa1, as well as those with inactivation of the NADH dehydrogenase Ndh51 or subunits of the mitochondrial pyruvate dehydrogenase complex, all display a defect in switching from an ovoid yeast to a filamentous cell morphology, which is a key aspect of virulence (1,46,57,87).…”

Genetic and Functional Investigation of Zn2Cys6Transcription Factors RSE2 and RSE3 in Podospora anserina

“…Mycelia were harvested, flash frozen in liquid nitrogen, and ground in a Fastprep apparatus (MP Bio) with glass beads and the RLT buffer provided with the RNA extraction kit (RNeasy plant kit [Qiagen]). For quantitative reverse transcription-PCR (RT-qPCR) analysis, 2 g of freshly extracted total RNA was reverse transcribed with oligo(dT) 15 using Superscript II reverse transcriptase (Invitrogen). The RT-qPCR amplification mixture (10 l) was examined with the LightCycler 480 SYBR green I master kit (Roche) on 2 l of a 1/10 dilution of the reverse transcription reaction.…”

“…Several studies indicate that the AOX is upregulated in oxidative stress conditions (14,15). It is developmentally regulated in the pathogenic dimorphic fungus Paracoccidioides brasiliensis (15) and plays an important role in the life cycle of the fungal plant pathogen Moniliophthora perniciosa (16). Recently, it has been shown that the AOX is relevant to the virulence of several human-pathogenic fungi, such as Cryptococcus neoformans (17), Aspergillus fumigatus (18), and Paracoccidioides brasiliensis (19).…”

“…It is believed to contribute to the maintenance of tricarboxylic acid (TCA) cycle turnover and to act as an overflow for electron transport, preventing the deleterious oxidative stress associated with the increased generation of mitochondrial reactive oxygen species (ROS). Several studies indicate that the AOX is upregulated in oxidative stress conditions (14,15). It is developmentally regulated in the pathogenic dimorphic fungus Paracoccidioides brasiliensis (15) and plays an important role in the life cycle of the fungal plant pathogen Moniliophthora perniciosa (16).…”