Polyamine transporters are involved in the uptake of salivary Histatin 5 in Candida albicans
Bajwa, Jashanjot Singh
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Hst 5 is a cationic peptide secreted in human saliva that has a prominent antifungal activity against C. albicans. Intracellular Hst 5 is shown to be required for antifungal activity. However mechanisms involved in the intracellular translocation of Hst 5 are not well understood. Intracellular transport of Hst 5 requires its initial binding to the candidal cell wall which is followed by its subsequent translocation across the plasma membrane to enter the cytosol. Translocation of Hst 5 across the plasma membrane being the key step in cytoslic translocation of Hst 5 is also responsible for its toxicity in C. albicans. Therefore it is important to understand the mechanism of intracellular translocation of Hst 5 to understand its mechanism of action. Polyamines are small polycationic molecules that are transported across plasma membrane using specific transmembrane proteins known as polyamine transporters. Agp2, Dur3, Sam3 and Gap1 are four major polyamine transporters in S. cerevisiae. These proteins are highly regulated proteins and are downregulated when cells are grown in the presence of high extracellular concentrations of polyamines to protect the cells from the toxic effects of excess polyamines. These transporters have been well studied in S. cerevisiae but are not well characterized in C. albicans. We hypothesize that Hst 5 being a small polycationic peptide may utilize polyamine transporters during its intracellular translocation in C. albicans. Objectives : To study the role of polyamine transporters in intracellular translocation of Hst 5 in C. albicans. Methods : Polyamine transporters in C. albicans were identified and characterized using homology studies and phylogenetic analysis. Deletion mutants for five polyamine transporter genes Dur3, Dur31, Dur33, Dur34 and Agp2 were constructed. Dur3 gene was reconstituted in Dur3 deletion mutants by complementation. Candidacidal activity of Hst 5 on these mutants was studied using microdilution candidacidal assays and uptake of Hst 5 was studied using confocal microscopy. Competition between Hst 5 and polyamines was studied using candidacidal assays and uptake assays. Additionally, Hst 5 killing and uptake was studied in cells grown in polyamines. Results : One homolog for S. cerevisiae Agp2 (ScAGP2) and multiple homologs for S. cerevisiae Dur3 (ScDur3) protein were found in C. albicans. There was no homolog of S. cerevisiae Sam3 in C. albicans. Cells treated with Hst 5 in the presence of polyamines exhibited a lower sensitivity to Hst 5 mediated killing due to reduced uptake of Hst 5 that might be a result of direct competition between two molecules for a common uptake pathway. Cells grown in polyamines also showed a reduced senstivity to Hst 5 killing due to reduced uptake of Hst 5. This reduced uptake might be due to down regulation of polyamine transporters that might be involved in Hst 5 uptake in the response to high extracellular concentrations of polyamines in the growth media. Polyamine transporter deletions were successfully constructed and gene deletion was confirmed with PCR. Dur3 deletion mutants showed reduced Hst 5 killing and uptake at all physiological concentrations of Hst 5. Agp2 deletion mutants showed reduced Hst 5 mediated killing only at lower Hst 5 concentrations. Reduced sensitivity to Hst 5 mediated killing was not seen in Dur31, Dur33 and Dur34 deletion mutants. Conclusions : Polyamine transporters are involved in Hst 5 uptake in C. albicans. Dur3 was found to play a major role in Hst 5 uptake while Agp2 plays only a minor role. Dur31, Dur33 and Dur34 transporters are not involved in Hst 5 uptake. Since intracellular Hst 5 is required for killing activity in C. albicans, this study will be helpful for understanding the mechanism of action of Hst 5 against C. albicans.