Studies on the relationship between pulsed UV light irradiation and the simultaneous occurrence of molecular and cellular damage in clinically-relevant Candida albicans

J Microbiol Methods. 2011 Feb;84(2):317-26. doi: 10.1016/j.mimet.2010.12.021. Epub 2010 Dec 23.

Abstract

This constitutes the first study to report on the relationship between pulsed UV light (PL) irradiation and the simultaneous occurrence of molecular and cellular damage in clinical strains of Candida albicans. Microbial protein leakage and propidium iodide (PI) uptake assays demonstrated significant increases in cell membrane permeability in PL-treated yeast that depended on the amount of UV pulses applied. This finding correlated well with the measurement of increased levels of lipid hydroperoxidation in the cell membrane of PL-treated yeast. PL-treated yeast cells also displayed a specific pattern of intracellular reactive oxygen species (ROS) generation, where ROS were initially localised in the mitochondria after low levels of pulsing (UV dose 0.82 μJ/cm(2)) before more wide-spread cytosolic ROS production occurred with enhanced pulsing. Intracellular ROS levels were measured using the specific mitochondrial peroxide stain dihydrorhodamine 123 and the cytosolic oxidation stain dichloroflurescin diacetate. Use of the dihydroethidium stain also revealed increased levels of intracellular superoxide as a consequence of augmented pulsing. The ROS bursts observed during the initial phases of PL treatment was consistent with the occurrence of apoptotic cells as confirmed by detection of specific apoptotic markers, abnormal chromatin condensation and externalisation of cell membrane lipid phosphatidylserine. Increased amount of PL-irradiation (ca. UV does 1.24-1.65 μJ/cm(2)) also resulted in the occurrence of late apoptotic and necrotic yeast phenotypes, which coincided with the transition from mitochondrial to cytosolic localisation of ROS and with irreversible cell membrane leakage. Use of the comet assay also revealed significant nuclear damage in similarly treated PL samples. Although some level of cellular repair was observed in all test strains during sub-lethal exposure to PL-treatments (≤20 pulses or UV dose 0.55 μJ/cm(2)), this was absent in similar samples exposed to increased amounts of pulsing. This study showed that PL-irradiation inactivates C. albicans test strains through a multi-targeted process with no evidence of microbial ability to support cell growth after ≤20 pulses. Implications of our findings in terms of application of PL for contact-surface disinfection are discussed.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis
  • Candida albicans / isolation & purification
  • Candida albicans / metabolism
  • Candida albicans / radiation effects*
  • Candidiasis / microbiology*
  • Cell Membrane / chemistry
  • Cell Membrane / radiation effects
  • Cell Membrane Permeability / radiation effects
  • Chromatin / metabolism
  • Comet Assay
  • Cytoplasm / chemistry
  • DNA Damage*
  • Humans
  • Lipid Peroxides / analysis
  • Microbial Viability / radiation effects*
  • Phosphatidylserines / analysis
  • Reactive Oxygen Species / analysis
  • Ultraviolet Rays*

Substances

  • Chromatin
  • Lipid Peroxides
  • Phosphatidylserines
  • Reactive Oxygen Species