The role of apoptosis on Candida albicans-induced oral epithelial cell death

Oropharyngeal candidiasis is the most common opportunistic fungal infection in immunosuppressed and immunocompromised populations. During this infection, interactions between Candida albicans (C. albicans ) and oral epithelial cells result in oral epithelial cell death. This is clinically manifested by the development of ulcerations on the oral mucosa generally associated with local discomfort and dysphagia, resulting in poor nutrition and further impacting the quality of life of patients. Apoptosis is a physiologic component of the development and homeostasis of multicellular organisms, but also occurs in response to stress conditions such as infections. Induction of apoptosis typically results in the activation of caspases, a group of cysteine proteases essential for progression of apoptotic pathways. In vitro studies have shown that C. albicans induces early apoptotic alterations in oral epithelial cells, however these studies have also shown that treatment of infected oral epithelial cells with caspase inhibitors does not prevent their death. The reasons for these contradictory results are unknown and it is still not clear if C. albicans fully stimulates oral epithelial signaling pathways that promote apoptotic cell death. Thus, the aims of this study are to i) characterize C. albicans -induced apoptotic morphological alterations in oral epithelial cells utilizing assays that detect early and late apoptotic events, and ii) investigate the activation of apoptotic signaling pathways and expression of anti- and pro-apoptotic genes in C. albicans-infected oral epithelial cells.;The ability of C. albicans to activate apoptotic signaling pathways and promote apoptotic alterations in oral epithelial cells was investigated using a human oral epithelial cell-C. albicans co-culture system. Immunostaining methods and TUNEL assay were used to assess phosphatidylserine exposure and genomic DNA fragmentation, respectively. Mitochondrial transmembrane potential was measured using the JC1 probe and the amount of cytochrome c in cytosolic and mitochondrial fractions was quantified by ELISA. Caspase activity was measured using caspase-fluorometric assays and PARP-1 integrity was monitored over time by Western blotting and an enzymatic assay. Finally, expression of key anti- and pro-apoptotic genes was measured using PCR arrays. Negative controls included uninfected epithelial cultures and C. albicans alone. Statistical significance was determined by two-tailed t-test, with p<0.05 considered significant.;Results show that C. albicans induces early apoptotic changes in oral epithelial cells. These early changes characterized by phosphatidylserine exposure to the outer layer of the plasma membrane were found in over 50% of oral epithelial cells. However, only 15% of the infected cells underwent mid-late apoptotic changes as measured by DNA degradation. These results suggest that only a small percentage of the infected cells presenting early apoptotic changes progress towards the late stages of apoptosis. At the molecular level, C. albicans caused a loss of the mitochondrial membrane potential and translocation of cytochrome c from the mitochondria into the cytosol of infected cells, suggesting that the intrinsic (mitochondrial) apoptotic pathway is activated in response to C. albicans. Translocation of cytochrome c into the cytosol is typically followed by the activation of caspase-3 and caspase-9. Caspase-3 and -9 activities increased during the first hours of infection. However at later times, their activities returned to levels similar to the ones observed in uninfected cells. Once activated, caspase-3 catalyzes the cleavage of PARP-1, a DNA repair enzyme. This is a key event during apoptosis, as it establishes the irreversible progression of apoptotic cell death pathways. PARP-1 was cleaved into non-apoptotic, necrotic-like fragments during infection. This suggests that necrotic proteases were also activated during C. albicans infection. Finally, 5 anti-apoptotic genes were significantly up-regulated and 2 pro-apoptotic genes were down-regulated in response to C. albicans. Altogether, these findings indicate that oral epithelial apoptotic pathways are initially activated in response to C. albicans, but fail to properly progress over time and promote apoptotic epithelial cell death.