Candida albicans Flu1 transporters and Cek1 MAPK signaling modulate the fungicidal activity of Hst 5
Candida albicans is the causative agent of oropharyngeal candidiasis (OPC), which is prevalent in immunocompromised patients, individuals with dry mouth, or patients with prolonged antibiotic therapies that reduce oral commensal bacteria. Human salivary histatins, including histatin 5 (Hst 5), are small cationic proteins that are the major source of fungicidal activity of saliva. Fungicidal activity of Hst 5 requires intracellular translocation and accumulation to a threshold concentration for it to disrupt cellular processes. Previously, we observed that total cytosolic levels of Hst 5 were gradually reduced from intact cells, suggesting that C. albicans possesses a transport mechanism for efflux of Hst 5. Since we identified C. albicans polyamine transporters responsible for Hst 5 uptake, we hypothesized that one or more polyamine efflux transporters may be involved in the efflux of Hst 5 ( Chapter 1 ). C. albicans FLU1 and TPO2 were found to be the closest homologs of Saccharomyces cerevisiae TPO1, which encodes a major spermidine efflux transporter, indicating that the products of these two genes may be involved in efflux of Hst 5. We found that flu1Δ/Δ cells, but not tpo2Δ/Δ cells, had significant reductions in their rates of Hst 5 efflux and had significantly higher cytoplasmic Hst 5 and Hst 5 susceptibilities than did the wild type. We also found that flu1Δ/Δ cells had reduced biofilm formation compared to wild-type cells in the presence of Hst 5. Transcriptional levels of FLU1 were not altered over the course of treatment with Hst 5; therefore, Hst 5 is not likely to induce FLU1 gene overexpression as a potential mechanism of resistance. Thus, Flu1, but not Tpo2, mediates efflux of Hst 5 and is responsible for reduction of its toxicity in C. albicans. Hsts are rapidly degraded in vivo, limiting their usefulness as therapeutic agents despite their lack of toxicity. In my study, I used a conjugate peptide of spermidine (Spd) linked to the active fragment of Hst 5 (Hst 5 4-15 ), based upon our findings that C. albicans spermidine transporters are required for Hst 5 uptake and fungicidal activity ( Chapter 2 ). We found that Hst 5 4-15 -Spd was significantly more effective in killing C. albicans than Hst 5 alone in both planktonic and biofilm growth and that Hst 5 4-15 -Spd retained high activity in both serum and saliva. In addition, C. albicans senses its environment by MAP Kinase activation that can also modulate the activity of some antifungal drugs including Hst 5. We found that phosphorylation of the MAPK Cek1, induced either by N-acetyl- glucosamine (GlcNAc) or serum, or its constitutive activation by deletion of its phosphatase Cpp1, increased C. albicans susceptibility to Hst 5 ( Chapter 3 ). Changes in fungal cell surface glycostructures also modulated Hst 5 sensitivity, and Cek1 inducing conditions resulted in a higher uptake rate of Hst 5.