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. 2014 Jul 15;5(4):e01334-14.
doi: 10.1128/mBio.01334-14.

Mechanisms underlying the exquisite sensitivity of Candida albicans to combinatorial cationic and oxidative stress that enhances the potent fungicidal activity of phagocytes

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Mechanisms underlying the exquisite sensitivity of Candida albicans to combinatorial cationic and oxidative stress that enhances the potent fungicidal activity of phagocytes

Despoina Kaloriti et al. mBio. .

Abstract

Immune cells exploit reactive oxygen species (ROS) and cationic fluxes to kill microbial pathogens, such as the fungus Candida albicans. Yet, C. albicans is resistant to these stresses in vitro. Therefore, what accounts for the potent antifungal activity of neutrophils? We show that simultaneous exposure to oxidative and cationic stresses is much more potent than the individual stresses themselves and that this combinatorial stress kills C. albicans synergistically in vitro. We also show that the high fungicidal activity of human neutrophils is dependent on the combinatorial effects of the oxidative burst and cationic fluxes, as their pharmacological attenuation with apocynin or glibenclamide reduced phagocytic potency to a similar extent. The mechanistic basis for the extreme potency of combinatorial cationic plus oxidative stress--a phenomenon we term stress pathway interference--lies with the inhibition of hydrogen peroxide detoxification by the cations. In C. albicans this causes the intracellular accumulation of ROS, the inhibition of Cap1 (a transcriptional activator that normally drives the transcriptional response to oxidative stress), and altered readouts of the stress-activated protein kinase Hog1. This leads to a loss of oxidative and cationic stress transcriptional outputs, a precipitous collapse in stress adaptation, and cell death. This stress pathway interference can be suppressed by ectopic catalase (Cat1) expression, which inhibits the intracellular accumulation of ROS and the synergistic killing of C. albicans cells by combinatorial cationic plus oxidative stress. Stress pathway interference represents a powerful fungicidal mechanism employed by the host that suggests novel approaches to potentiate antifungal therapy. Importance: The immune system combats infection via phagocytic cells that recognize and kill pathogenic microbes. Human neutrophils combat Candida infections by killing this fungus with a potent mix of chemicals that includes reactive oxygen species (ROS) and cations. Yet, Candida albicans is relatively resistant to these stresses in vitro. We show that it is the combination of oxidative plus cationic stresses that kills yeasts so effectively, and we define the molecular mechanisms that underlie this potency. Cations inhibit catalase. This leads to the accumulation of intracellular ROS and inhibits the transcription factor Cap1, which is critical for the oxidative stress response in C. albicans. This triggers a dramatic collapse in fungal stress adaptation and cell death. Blocking either the oxidative burst or cationic fluxes in human neutrophils significantly reduces their ability to kill this fungal pathogen, indicating that combinatorial stress is pivotal to immune surveillance.

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Figures

FIG 1
FIG 1
Combinatorial cationic and oxidative stress kills evolutionarily divergent yeasts. Candida albicans (CA372) (A), Candida glabrata (ATCC 2001) (B), Saccharomyces cerevisiae (BY4741) (C), and Schizosaccharomyces pombe (972) (D) (see Table S1 in the supplemental material) were grown to exponential phase and then exposed to stress for 4 h, and cell death was quantified by propidium iodide staining and FACS analysis for no stress, 5 mM H2O2, 1 M NaCl, or 1M NaCl plus 5 mM H2O2. Data are means standard deviations (C. albicans, n = 3; C. glabrata, n = 4; S. cerevisiae, n = 3; S. pombe, n = 4).
FIG 2
FIG 2
Cationic and oxidative stresses act synergistically to kill C. albicans. First, the dose-dependent impact of each individual stress upon C. albicans viability was determined by propidium iodide staining and FACS analysis. Using mathematical modeling, we then normalized the potency of each stress (e.g., DNaCl) relative to the dose required to achieve 100% killing [(DX)NaCl]. The impacts of different combinations of cationic plus oxidative stress were then experimentally determined, and the data were plotted in this normalized isobologram. If the two stresses acted in an additive fashion, the data points would lie on the diagonal between 0.1 and 1.0 (dotted line). The points lie well below this line, indicating formally that cationic and oxidative stresses act synergistically to kill C. albicans.
FIG 3
FIG 3
Transcript profiling of C. albicans following exposure to individual and combinatorial cationic and oxidative stresses. C. albicans (CA372) cells exposed to 1 M NaCl, 5 mM H2O2, or a combination of these stresses were subjected to microarray analysis. (A) Venn diagram presenting the numbers of genes that were consistently upregulated by ≥2.5-fold in three independent experiments. The microarray findings were validated by qRT-PCR analysis of individual transcripts (HGT10, GPD2, TRR1, and CAT1); their levels were quantified relative to the internal ACT1 mRNA control. Data are means standard deviations (n = 3). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) GO terms that were significantly enriched in the subsets of genes upregulated by the individual and combinatorial stresses. The microarray data set is presented in Table S2 in the supplemental material and are also available at ArrayExpress (http://www.ebi.ac.uk/arrayexpress/experiments/E-MEXP-3003/) under accession number E-MEXP-3003.
FIG 4
FIG 4
Impact of combinatorial cationic plus oxidative stress upon Hog1. C. albicans cells were exposed to 5 mM H2O2, 1 M NaCl, or 5 mM H2O2 plus 1 M NaCl. (A) Hog1 phosphorylation status in C. albicans (ML258) cells during exposure to stress, as revealed by Western blotting with a phospho-specific antibody. (B) Cellular localization of Hog1-YFP in C. albicans (JC63) cells determined by fluorescence microscopy before (no stress) and after 10 min of stress. The positions of nuclei were determined by DAPI staining.
FIG 5
FIG 5
Impact of combinatorial cationic plus oxidative stress upon Cap1. C. albicans cells were exposed to 5 mM H2O2, 1 M NaCl, or 5 mM H2O2 plus 1 M NaCl. (A) Western blotting with an anti-Cap1 antibody to examine the gel shifts associated with Cap1 phosphorylation in C. albicans (JC948) cells after 10 min of stress. (Phosphatase controls are shown in Fig. S3 in the supplemental material.) (B) Localization of Cap1-GFP in C. albicans (JC1060) cells via fluorescence microscopy after 10 min of stress. DAPI staining revealed the positions of nuclei.
FIG 6
FIG 6
Catalase drives H2O2 detoxification and is inhibited by cations. (A) The detoxification of extracellular H2O2 by C. albicans cells is dependent on catalase. Data are means standard deviations (SD) for wild type (WT, CA372; n = 5); cat1 (n = 4), cap1 (n = 4), and hog1 (n = 3) (see Table S1 in the supplemental material). The starting concentration was 5 mM H2O2 (100%). (B) NaCl and KCl (but not sorbitol) inhibited catalase activity in wild-type C. albicans cells (CA372). Data are means SD (n = 3). Catalase activity is expressed relative to levels in the untreated controls.
FIG 7
FIG 7
Ectopic catalase expression suppresses the elevated intracellular ROS levels and synergistic killing caused by combinatorial cationic plus oxidative stress. (A) Catalase activity was reduced in C. albicans wild-type (RM1000) cells following 60 min of combinatorial stress but was partially restored by ectopic catalase expression via ACT1-CAT1. Data are means standard deviations (n = 4) (see Table S1 in the supplemental material). (D) Intracellular ROS accumulation, assayed via dihydroethidium (DHE) fluorescence, was increased in C. albicans wild-type (RM1000) cells exposed to combinatorial stress for 60 min but was reduced in ACT1-CAT1 cells (n = 5). (E) Ectopic catalase expression suppressed the synergistic killing of C. albicans cells by combinatorial oxidative plus cationic stress. Cell death was quantified in wild-type (RM1000) and ACT1-CAT1 cells by propidium iodide staining and FACS analysis (n = 3). P values between 0.05 and 0.001 were considered: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
FIG 8
FIG 8
Phagocytic killing of C. albicans cells is dependent on cationic fluxes as well as the oxidative burst. (A) Killing of C. albicans (CA372) by human neutrophils was significantly decreased by drugs that inhibited the oxidative burst (apocynin) and cationic fluxes (glibenclamide). Data are means standard deviations (n = 8). Neutrophils were isolated from human blood and treated with 200 µM glibenclamide and/or apocynin. C. albicans cells were grown to exponential phase and incubated with neutrophils for 2 h at a ratio of 1:10 (C. albicans:neutrophils). (B) Neutrophils from p47phox−/− mice did not kill C. albicans (CA372) efficiently (n = 4). Neutrophils were isolated from the bone marrow of wild-type and p47phox−/− knockout mice, which lack NADPH oxidase activity. Exponentially growing C. albicans cells were incubated with these neutrophils for 2 h at a ratio of 1:10. P values between 0.05 and 0.001 were considered: *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.
FIG 9
FIG 9
Impact of combinatorial stress on oxidative stress adaptation. When C. albicans cells are exposed to an oxidative stress, Cap1 mediates the activation of catalase and other oxidative stress genes, leading to ROS detoxification, stress adaptation, and survival (left panel). However, when C. albicans cells are exposed to combinatorial stress, for example, during phagocytosis by neutrophils, the cations inhibit catalase, thereby reducing H2O2 detoxification and leading to intracellular ROS accumulation. This prevents the activation of Cap1, leading to a precipitous collapse in oxidative stress adaptation and cell death. Cations may also affect Cap1 activity by other ROS-independent mechanisms (see the text).

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References

    1. Odds FC. 1988. Candida and Candidiasis. London, United Kingdom
    1. Calderone RA, Clancy CJ. 2011. Candida and candidiasis. ASM Press, Washington, DC
    1. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci. Transl. Med. 4:165rv13. 10.1126/scitranslmed.30044040 - DOI - PubMed
    1. Nagao M. 2013. A multicentre analysis of epidemiology of the nosocomial bloodstream infections in Japanese university hospitals. Clin. Microbiol. Infect. 19:852–858. 10.1111/1469-0691.12083 - DOI - PubMed
    1. Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. 2004. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin. Infect. Dis. 39:309–317. 10.1086/421946 - DOI - PubMed

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