Integrative Model of Oxidative Stress Adaptation in the Fungal Pathogen Candida albicans

Komalapriya, Chandrasekaran; Kaloriti, Despoina; Tillmann, Anna; Yin, Zhikang; Herrero‐de‐Dios, Carmen; Jacobsen, Martin; Belmonte, Rodrigo; Cameron, Gary; Haynes, Ken; Grebogi, Celso; Moura, Arnaldo Vieira; Gow, Neil A. R.; Thiel, Marco; Quinn, Janet; Brown, Alistair J. P.; Romano, M. Carmen

doi:10.1371/journal.pone.0137750

Cited by 61 publications

(46 citation statements)

References 80 publications

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“…albicans (approximately 1.5 x 10 5 molecules per cell [33]). In bacteria, catalase overexpression has been reported to affect the requirement for iron [50].…”

Section: Resultsmentioning

confidence: 99%

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron

et al. 2017

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Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.

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“…albicans (approximately 1.5 x 10 5 molecules per cell [33]). In bacteria, catalase overexpression has been reported to affect the requirement for iron [50].…”

Section: Resultsmentioning

confidence: 99%

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron

et al. 2017

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“…Subsequently, additional PubMed queries were performed using a combination of the following key words: 1) Candida albicans, qPCR, gene expression; 2) Candida albicans, specific gene name of genes listed in Table 1. The following publications were selected in which the authors described the primers and cited when these primers were target-specific for the chosen virulence genes (Alves et al, 2014;Chen and Lan, 2015;Dai et al, 2013;Green et al, 2004;Hnisz et al, 2012;Komalapriya et al, 2015;Martchenko et al, 2004;Naglik et al, 2003b;Nailis et al, 2010;Zhu et al, 2011). Next, all primers were subjected to a step wise validation approach described below.…”

Section: Methodsmentioning

confidence: 99%

A quest to find good primers for gene expression analysis of Candida albicans from clinical samples

Alonso

Pavarina

Sousa

et al. 2018

Journal of Microbiological Methods

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Biofilm production contributes to several human diseases, including oral candidiasis. Among the Candida species, Candida albicans is the most prevalent. The expression of virulence genes is implicated in the pathogenic potential of Candida biofilms. However, the evaluation of microbial gene expression from in vivo biofilm samples is not trivial, specifically, assessment via quantitative PCR (qPCR) can be a challenge because of several species present in clinical samples. Hence, the necessity of primers specificity. The aim of this study was to evaluate through in silico and in vitro analyses the specificity of published primers and newly designed primers for C. albicans virulence genes: ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, SAP4, SOD1, SOD5 and ACT1 (normalizing gene). In silico analysis was performed through a PubMed search of articles with primer sequences that evaluated gene expression of C. albicans. Then, the sequence similarity of twenty-eight primers was checked through BLASTn and ClustalW2. The analysis of secondary structures was performed using mfold. When the primers did not present satisfactory characteristics (absence of secondary structures, not discrepant Tm of forward and reverse sequences and specificity) following in vitro analysis (i.e., end point PCR), new primers were designed using Beacon Designer™ and sequences obtained from the "Candida Genome Database". The selected primers were tested in vitro by end point PCR using a panel of genomic DNA from five different Candida species (C. albicans, Candida glabrata, Candida dubliniensis, Candida krusei, and Candida tropicalis). The resulting PCR products were visualized on agarose gel. qPCR reactions were performed to determine primers' optimal concentration and PCR efficiency. End point PCR demonstrated that published primers for the SAP1 and HWP1 were specific for C. albicans and the one for SOD1 reacted with C. albicans and C. dubliniensis. The sequence of primers designed for ACT1, ALS1 and HWP1 genes were specific for C. albicans, while the ones for CAP1, CAT1, EFG1, LIP3, and PLB1 were detected in C. albicans and C. dubliniensis. After optimization, all primers presented a single peak on melt curves, correlation coefficient of ≅1 and qPCR reaction efficiency of 90-110%, with slope of ≅-3.3. Therefore, these primers should be suitable for future gene expression analyses from clinical samples.

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“…Further, Cap1p, which is homologous to Saccharomyces cerevisiae Yap1p, is responsible for the activation of genes related to oxidative stress. When ROS accumulates, Cap1p oxidizes and accumulates in the nucleus, further activating genes carrying Cap1 response elements in their promoters 13 . However, Yap1p is not directly oxidized, but needs a glutathione peroxidase (Gpx)-like protein to transduce Yap1p signaling by creating a disulfide bond between Gpx3p and Cys36 (evolutionarily conserved redox-sensitive residues) and Yap1p with Cys598 14 .…”

Section: Discussionmentioning

confidence: 99%

Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage

Saibabu

Singh

Ansari

et al. 2017

Rev. Soc. Bras. Med. Trop.

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Introduction: Citronellal (Cit) possesses antifungal activity and has possible implications for reactive oxygen species (ROS) generation in Candida albicans. In this study, the effects of Cit on ROS generation and the mechanisms by which Cit exerts anti-Candida effects were examined. Methods: A 2′,7′-dichlorodihydrofluorescein diacetate assay was used to assess oxidative damage. Cell necrosis was determined by flow cytometry after FITC-Annexin V staining. Mitochondrial function was studied based on mitochondrial potential, metabolic activity (MTT assay), and phenotypic susceptibility on a non-fermentable carbon source. Membrane intactness and DNA damage were estimated by a propidium iodide (PI) uptake assay and 4',6-diamidino-2-phenylindole (DAPI) staining. Results: ROS generation was enhanced in response to Cit, leading to necrosis (2%). Additional hallmarks of cell death in response to Cit, such as mitochondrial membrane depolarization and DNA damage, were also observed. Cit treatment resulted in dysfunctional mitochondria, as evidenced by poor labeling with the mitochondrial membrane potential-sensitive probe rhodamine B, reduced metabolic activity (61.5%), and inhibited growth on a non-fermentable carbon source. Furthermore, Cit induced DNA damage based on DAPI staining. These phenotypes were reinforced by RT-PCR showing differences in gene expression (30-60%) between control and Cit-treated cells. Finally, PI uptake in the presence of sodium azide confirmed non-intact membranes and suggested that Cit activity is independent of the energy status of the cell. Conclusions: Cit possesses dual anticandidal mechanisms, including membrane-disruptive and oxidative damage. Taken together, our data demonstrated that cit could be used as a prominent antifungal drug.

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Integrative Model of Oxidative Stress Adaptation in the Fungal Pathogen Candida albicans

Cited by 61 publications

References 80 publications

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron

Elevated catalase expression in a fungal pathogen is a double-edged sword of iron

A quest to find good primers for gene expression analysis of Candida albicans from clinical samples

Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage

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