The Cch1-Mid1 High-Affinity Calcium Channel Contributes to the Virulence of Cryptococcus neoformans by Mitigating Oxidative Stress

Pathogenic fungi have developed mechanisms to cope with stresses imposed by hosts. For Cryptococcus spp., this implies active defense mechanisms that attenuate and ultimately overcome the onslaught of oxidative stresses in macrophages. Among cellular pathways within Cryptococcus neoformans'' arsenal is the plasma membrane high-affinity Cch1-Mid1 calcium (Ca²⁺) channel (CMC). Here we show that CMC has an unexpectedly complex and disparate role in mitigating oxidative stress. Upon inhibiting the Ccp1-mediated oxidative response pathway with antimycin, strains of C. neoformans expressing only Mid1 displayed enhanced growth, but this was significantly attenuated upon H₂O₂ exposure in the absence of Mid1, suggesting a regulatory role for Mid1 acting through the Ccp1-mediated oxidative stress response. This notion is further supported by the interaction detected between Mid1 and Ccp1 (cytochrome c peroxidase). In contrast, Cch1 appears to have a more general role in promoting cryptococci survival during oxidative stress. A strain lacking Cch1 displayed a growth defect in the presence of H₂O₂ without BAPTA [(1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, cesium salt] or additional stressors such as antimycin. Consistent with a greater contribution of Cch1 to oxidative stress tolerance, an intracellular growth defect was observed for the cch1Δ strain in the macrophage cell line J774A.1. Interestingly, while the absence of either Mid1 or Cch1 significantly compromises the ability of C. neoformans to tolerate oxidative stress, the absence of both Mid1 and Cch1 has a negligible effect on C. neoformans growth during H₂O₂ stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC.     

Adaptation of the Phytopathogenic Fungus Fusarium decemcellulare to Oxidative Stress

The adaptive response of the phytopathogenic fungus Fusarium decemcellulare to the oxidative stress induced by hydrogen peroxide and juglone (5-hydroxy-1,4-naphthoquinone) was studied. At concentrations higher than 1 mM, H₂O₂ and juglone completely inhibited the growth of the fungus. The 60-min pretreatment of logarithmic-phase cells with nonlethal concentrations of H₂O₂ (0.25 mM) and juglone (0.1 mM) led to the development of a resistance to high concentrations of these oxidants. The stationary-phase cells were found to be more resistant to the oxidants than the logarithmic-phase cells. The adaptation of fungal cells to H₂O₂ and juglone was associated with an increase in the activity of cellular catalase and superoxide dismutase, the main enzymes involved in the defense against oxidative stress.     

The catalytic subunit of DNA-dependent protein kinase is required for cellular resistance to oxidative stress independent of DNA double-strand break repair

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) are the two major kinases involved in DNA double-strand break (DSB) repair, and are required for cellular resistance to ionizing radiation. Whereas ATM is the key upstream kinase for DSB signaling, DNA-PKcs is primarily involved in DSB repair through the nonhomologous end-joining (NHEJ) mechanism. In addition to DSB repair, ATM has been shown to be involved in the oxidative stress response and could be activated directly in vitro on hydrogen peroxide (H₂O₂) treatment. However, the role of DNA-PKcs in cellular response to oxidative stress is not clear. We hypothesize that DNA-PKcs may participate in the regulation of ATM activation in response to oxidative stress, and that this regulatory role is independent of its role in DNA double-strand break repair. Our findings reveal that H₂O₂ induces hyperactivation of ATM signaling in DNA-PKcs-deficient, but not Ligase 4-deficient cells, suggesting an NHEJ-independent role for DNA-PKcs. Furthermore, DNA-PKcs deficiency leads to the elevation of reactive oxygen species (ROS) production, and to a decrease in cellular survival against H₂O₂. For the first time, our results reveal that DNA-PKcs plays a noncanonical role in the cellular response to oxidative stress, which is independent from its role in NHEJ. In addition, DNA-PKcs is a critical regulator of the oxidative stress response and contributes to the maintenance of redox homeostasis. Our findings reveal that DNA-PKcs is required for cellular resistance to oxidative stress and suppression of ROS buildup independently of its function in DSB repair.     

Neuroprotective Effects of Farnesene Against Hydrogen Peroxide-Induced Neurotoxicity In vitro

Oxidative stress is highly damaging to cellular macromolecules and is also considered a main cause of the loss and impairment of neurons in several neurodegenerative disorders. Recent reports indicate that farnesene (FNS), an acyclic sesquiterpene, has antioxidant properties. However, little is known about the effects of FNS on oxidative stress-induced neurotoxicity. We used hydrogen peroxide (H₂O₂) exposure for 6 h to model oxidative stress. Therefore, this experimental design allowed us to explore the neuroprotective potential of different FNS isomers (α-FNS and β-FNS) and their mixture (Mix-FNS) in H₂O₂-induced toxicity in newborn rat cerebral cortex cell cultures for the first time. For this aim, both MTT and lactate dehydrogenase assays were carried out to evaluate cell viability. Total antioxidant capacity (TAC) and total oxidative stress (TOS) parameters were used to assess oxidative alterations. In addition to determining of 8-hydroxy-2-deoxyguanosine (8-OH-dG) levels in vitro, the comet assay was also performed for measuring the resistance of neuronal DNA to H₂O₂-induced challenge. Our results showed that survival and TAC levels of the cells decreased, while TOS, 8-OH-dG levels and the mean values of the total scores of cells showing DNA damage (comet assay) increased in the group treated with H₂O₂ alone. But pretreatment of FNS suppressed the cytotoxicity, genotoxicity and oxidative stress, which were increased by H₂O₂ in clear type of isomers and applied concentration-dependent manners. The order of antioxidant effectiveness for modulating H₂O₂-induced oxidative stress-based neurotoxicity and genotoxicity is as β-FNS > Mix-FNS > α-FNS.     

Identification of Candida albicans heat shock proteins and Candida glabrata and Candida krusei enolases involved in the response to oxidative stress

In the past two decades, Candida species have become the second leading cause of invasive mycosis in immunocompromised patients. In order to colonize their hosts, these microorganisms express adhesins and cell wall proteins that allow them to adhere and neutralize the reactive oxygen species produced by phagocytic cells during the respiratory burst. However, the precise mechanism by which Candida cell wall proteins change their expression in response to oxidative stress has not been described. In an attempt to understand this change in response to oxidative stress, in this study, three Candida species, namely, C. albicans, C. glabrata and C. krusei, were exposed to increasing concentrations of H₂O₂ and induced cell wall proteins were identified by two-dimensional gel electrophoresis and peptide mass fingerprinting. Sequence analysis of differential proteins led to the identification of two heat-shock proteins in C. albicans, two enolases in C. glabrata and one enolase in C. krusei. Enolases may be involved in the protection of pathogenic cells against oxidative stress as suggested by the decrease in their expression when they were exposed to high concentrations of H₂O₂. To our knowledge, this is the first demonstration that expression of these proteins changes in response to oxidative stress in different Candida species. This knowledge can eventually facilitate both an early diagnosis and a more efficient treatment of this mycosis.     

The Calcium Channel Blocker Verapamil Inhibits Oxidative Stress Response in Candida albicans

Candida albicans is a common opportunistic fungal pathogen, causing both superficial candidiasis and life-threatening systemic infections in immune-compromised individuals. Calcium signaling is responsible for this pathogen in responding to several stresses, such as antifungal drugs, alkaline pH and membrane-perturbing agents. Our recent study revealed that it is also involved in oxidative stress response. In this study, we investigated the effect of verapamil, an L-type voltage-gated calcium channel blocker, on oxidative stress response in this fungus. The addition of verapamil resulted in increased sensitivity to the oxidative agent H₂O₂, which is associated with a decrease of calcium fluctuation under the stress. Moreover, this agent caused enhanced oxidative stress, with increased levels of ROS and enhanced dysfunction of the mitochondria under the oxidative stress. Further investigations in SOD activity, GSH contents and expression of oxidative stress response-related genes indicated that the effect of verapamil is related to the repression of oxidative stress response. Our findings demonstrated that verapamil has an inhibitory effect on oxidative stress response, confirming the relationship between calcium signaling and oxidative stress in C. albicans. Therefore, calcium channels may be potential targets for therapy to enhance the efficacy of oxidative stress against C. albicans-related infections.     

Respiratory Activity and Naphthoquinone Synthesis in the Fungus Fusarium decemcellulare Exposed to Oxidative Stress

The effect of oxidants (hydrogen peroxide and juglone) on the growth, respiration, and naphthoquinone synthesis in the fungus Fusarium decemcellulare was studied. The addition of the oxidants to the exponential-phase fungus inhibited cell respiration (either partially or completely, depending on the oxidant concentration), culture growth, and naphthoquinone synthesis. The treatment of fungal cells with nonlethal concentrations of H₂O₂ (below 0.25 mM) and juglone (below 0.1 mM) induced the resistance of cell respiration to cyanide. The residual respiration in the presence of cyanide could be inhibited by benzohydroxamic acid, indicating the occurrence of alternative oxidase. Increased concentrations of oxidants (0.25 mM juglone and 0.5 mM H₂O₂) rapidly and irreversibly inhibited cell respiration. These observations suggest that the mitochondrial respiratory chain of fungal cells exposed to oxidative stress is subject to the action of active oxygen species. The treatment of fungal cells with nonlethal concentrations of H₂O₂ and juglone activated cellular glutathione reductase and glucose-6-phosphate dehydrogenase, which are protective enzymes against oxidative stress.     

Quorum sensing enhancement of the stress response promotes resistance to quorum quenching and prevents social cheating

Quorum sensing (QS) coordinates the expression of virulence factors and allows bacteria to counteract the immune response, partly by increasing their tolerance to the oxidative stress generated by immune cells. Despite the recognized role of QS in enhancing the oxidative stress response, the consequences of this relationship for the bacterial ecology remain unexplored. Here we demonstrate that QS increases resistance also to osmotic, thermal and heavy metal stress. Furthermore a QS-deficient lasR rhlR mutant is unable to exert a robust response against H₂O₂ as it has less induction of catalase and NADPH-producing dehydrogenases. Phenotypic microarrays revealed that the mutant is very sensitive to several toxic compounds. As the anti-oxidative enzymes are private goods not shared by the population, only the individuals that produce them benefit from their action. Based on this premise, we show that in mixed populations of wild-type and the mexR mutant (resistant to the QS inhibitor furanone C-30), treatment with C-30 and H₂O₂ increases the proportion of mexR mutants; hence, oxidative stress selects resistance to QS compounds. In addition, oxidative stress alone strongly selects for strains with active QS systems that are able to exert a robust anti oxidative response and thereby decreases the proportion of QS cheaters in cultures that are otherwise prone to invasion by cheats. As in natural environments stress is omnipresent, it is likely that this QS enhancement of stress tolerance allows cells to counteract QS inhibition and invasions by social cheaters, therefore having a broad impact in bacterial ecology.     

Cellular Responses of Candida albicans to Phagocytosis and the Extracellular Activities of Neutrophils Are Critical to Counteract Carbohydrate Starvation,Oxidative and Nitrosative Stress

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO^•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.