IADR Abstract Archives
Amino-acid Starvation and DNA-protein-crosslink Repair Affect Candida Oxidative Stress Susceptibility
Objectives: Because oxidative stress is an important antimicrobial mechanism of phagocytes, this study aimed to investigate the effects of amino-acid starvation condition that mimics intraphagosomal environment and of DNA-protein-crosslink (DPC) repair on Candida albicans sensitivity to oxidative stress and neutrophil killing.
Methods: Growth rates of Candida albicans strains, including wild type (SC5314), DPC repair protease null mutant (Cawss1-/-), homologous recombination gene null mutant (Carad52-/-), and double deletion (Cawss1-/-rad52-/-) were determined. All were grown in Yeast-Peptone-Dextrose (YPD) media overnight and then switched to YNBA with and without amino acid supplements in the presence or absence of hydrogen peroxide (H2O2) for 24 hours at 30°C. OD600 was measured hourly using Multiskan SkyHigh Microplate Spectrophotometer. For neutrophil killing assay, neutrophils were freshly isolated from healthy volunteers using PolymorphPrep in RPMI1640. The study protocol was approved by the Faculty IRB (HREC-DCU 2011-124). Log phase C. albicans was incubated with heat-inactivated serum before being co-cultured with neutrophils for two hours. After cell lysis, surviving Candida were recovered on YPD plates for CFU counts. Percentage of neutrophil-killing was calculated by comparing CFU counts to no-neutrophil control.
Results: Based on growth rates, in normal laboratory condition with amino acid supplements, while all strains showed sensitivity to H2O2, the mutant strains showed higher sensitivity than wildtype. Interestingly, amino-acid starvation decreased fungal sensitivity to H2O2 in wild type, but not in the mutants. Neutrophil killing assays indicated that, compared to the wildtype, all mutant strains were more susceptible to neutrophil killing with Cawss1-/-, Carad52-/-, and Cawss1-/-rad52-/- being 1.22± 0.35, 1.10±0.19, and 1.22±0.41 times more susceptible than wild type, respectively.
Conclusions: Amino-acid starvation affects the growth rate of C. albicans and decreased the sensitivity of C.albicans to oxidative stress. Disruption of DPC repair genes render C. albicans more susceptible to oxidative stress and to killing by neutrophils.
Methods: Growth rates of Candida albicans strains, including wild type (SC5314), DPC repair protease null mutant (Cawss1-/-), homologous recombination gene null mutant (Carad52-/-), and double deletion (Cawss1-/-rad52-/-) were determined. All were grown in Yeast-Peptone-Dextrose (YPD) media overnight and then switched to YNBA with and without amino acid supplements in the presence or absence of hydrogen peroxide (H2O2) for 24 hours at 30°C. OD600 was measured hourly using Multiskan SkyHigh Microplate Spectrophotometer. For neutrophil killing assay, neutrophils were freshly isolated from healthy volunteers using PolymorphPrep in RPMI1640. The study protocol was approved by the Faculty IRB (HREC-DCU 2011-124). Log phase C. albicans was incubated with heat-inactivated serum before being co-cultured with neutrophils for two hours. After cell lysis, surviving Candida were recovered on YPD plates for CFU counts. Percentage of neutrophil-killing was calculated by comparing CFU counts to no-neutrophil control.
Results: Based on growth rates, in normal laboratory condition with amino acid supplements, while all strains showed sensitivity to H2O2, the mutant strains showed higher sensitivity than wildtype. Interestingly, amino-acid starvation decreased fungal sensitivity to H2O2 in wild type, but not in the mutants. Neutrophil killing assays indicated that, compared to the wildtype, all mutant strains were more susceptible to neutrophil killing with Cawss1-/-, Carad52-/-, and Cawss1-/-rad52-/- being 1.22± 0.35, 1.10±0.19, and 1.22±0.41 times more susceptible than wild type, respectively.
Conclusions: Amino-acid starvation affects the growth rate of C. albicans and decreased the sensitivity of C.albicans to oxidative stress. Disruption of DPC repair genes render C. albicans more susceptible to oxidative stress and to killing by neutrophils.